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Year: 2012
Role of aldosterone in potassium homeostasis and pregnancy
Abhijeet Pandurang Todkar
Posted at the Zurich Open Repository and Archive, University of Zurich
http://dx.doi.org/10.5167/uzh-73868
Originally published at:
Pandurang Todkar, Abhijeet. Role of aldosterone in potassium homeostasis and pregnancy. 2012, University
of Zurich, Faculty of Medicine.
Role of Aldosterone in Potassium Homeostasis and Pregnancy
Dissertation
zur
Erlangung der naturwissenschaftlichen Doktorwürde
(Dr. sc. nat.)
vorgelegt der
Mathematisch-naturwissenschaftlichen Fakultät
der
Universität Zürich
von
Abhijeet Pandurang Todkar
aus Indien
Promotionskomitee
Prof. Dr. med. Carsten A. Wagner (Vorsitz und Leitung der Dissertation)
Prof. Dr. med. Johannes Loffing (Leitung der Dissertation)
Prof. Dr. med. François Verrey
Prof. Dr. med. Dominique Eladari
Zürich, 2012
Contents
SUMMARY ........................................................................................................................... 5
DEUTSCHE ZUSAMMENFASSUNG DER DOKTORARBEIT .............................................. 9
1 INTRODUCTION: ............................................................................................................ 14
Aldosterone......................................................................................................................... 14
Aldosterone synthase (Cyp11b2)..................................................................................... 15
1.1 Project 1. Role of aldosterone in renal potassium excretion .......................................... 16
1.1.1 Aldosterone synthesis and regulation ..................................................................... 16
1.1.2 Target tissues and specificity for the receptors ....................................................... 16
1.1.3 Potassium homeostasis - External and internal K+ balance .................................... 17
1.1.4 Potassium handling in the kidney............................................................................ 19
1.1.5 Potassium secretion - Critical role of the ASDN ...................................................... 20
1.1.6 Structure, function and regulation of sodium and potassium channels and transporter
in the ASDN..................................................................................................................... 23
1.1.6.1 The epithelial sodium channel (ENaC) ............................................................. 23
1.1.6.2 ROMK .............................................................................................................. 26
1.1.6.3 BK or Maxi K channels ..................................................................................... 27
1.1.6.4 NCC ................................................................................................................. 28
1.1.7 Potassium handling by the colon ............................................................................ 30
1.2 Project 2. Role of aldosterone in pregnancy .................................................................. 31
1.2.1 Pregnancy specific disorder- Pre-eclampsia ........................................................... 31
1.2.2 Pregnancy specific disorder- IUGR ......................................................................... 31
1.2.3 Abnormal placental implantation - common to pre-eclampsia and IUGR................. 32
1.2.4 IUGR and pre-eclampsia may have different pathophysiologies ............................. 33
1.2.5 Placental efficiency- an important factor for fetal growth ......................................... 34
1.2.6 Role of angiogenic and antiangiogenic, and vasoactive factors in the development of
pre-eclampsia .................................................................................................................. 35
1.2.7 Possible role of aldosterone in the development of the pre-eclamapsia .................. 38
1.2.8 Different components of aldosterone effector mechanism expressed in placenta ... 39
1.2.9 High salt diet during pregnancy may affect blood pressure and pregnancy outcome
........................................................................................................................................ 40
2 AIMS OF THE STUDY ..................................................................................................... 41
2.1 Role of aldosterone in renal potassium excretion....................................................... 41
2.2 Role of aldosterone in pregnancy .............................................................................. 41
3 MATERIAL AND METHODS ............................................................................................ 42
3.1 Role of aldosterone in renal potassium excretion....................................................... 42
2
3.1.1 Animal model ...................................................................................................... 42
3.1.2 Metabolic cage experiments ................................................................................ 42
3.1.3 Experimental protocol for dietary potassium loading ........................................... 42
3.1.4 Changes in tubular workload ............................................................................... 43
3.1.5 Role of angiotensin II in renal adaptation to high potassium diet ......................... 43
3.1.6 Analysis of the urinary concentration capability of AS+/+ and AS-/- mice ............... 44
3.1.7 RNA extraction from the kidney ........................................................................... 44
3.1.8 Quantitative Real-time PCR ................................................................................ 45
3.1.9 Membrane preparation and western blot analysis ............................................... 46
3.1.10 Immunohistochemistry ...................................................................................... 47
3.1.11 Ussing chamber experiments ............................................................................ 48
3.1.12 Statistical analysis ............................................................................................. 48
3.2. Role of aldosterone in pregnancy ............................................................................. 49
3.2.1 Animals ............................................................................................................... 49
3.2.2 Study design and treatment ................................................................................ 49
3.2.3 Tail cuff blood pressure measurements ............................................................... 50
3.2.4 Urinary protein concentration measurement ........................................................ 50
3.2.5 RNA extraction .................................................................................................... 50
3.2.6 Semi-quantitative Real-time PCR ........................................................................ 51
3.2.7 Histology ............................................................................................................. 52
3.2.8 Immunoblotting ................................................................................................... 52
3.2.9 Statistical analysis ............................................................................................... 53
4 RESULTS ........................................................................................................................ 54
4.1 Aldosterone dependent and independent regulation of renal K+-excretion ................. 54
4.1.1 Response to high K+ loading ............................................................................... 54
4.1.2 Effect of high K+ diet on distal nephron K+ and Na+ channels: Increased apical
expression of ROMK and maintained ENaC cleavage in AS-/- mice on 2% K+ diet ....... 56
4.1.3 Functional ENaC and apical localization of ENaC in the late DCT in AS-/- mice on
2% K+ diet .................................................................................................................... 64
4.1.4 Impaired activation of colonic Na+ and K+ channels in AS-/- during 2% K+ loading 64
4.1.5 Role of angiotensin II in AS-/- mice....................................................................... 66
4.1.6 Decreased sodium chloride co-transporter (NCC) protein expression and activity in
AS-/- mice on 2% K+ diet ............................................................................................... 68
4.1.7 Similar deoxycoticosterone levels in AS-/- mice on control and 2% K+ diet ........... 68
4.1.8 AS-/- mice can concentrate urine similar to AS+/+ mice ......................................... 68
4.2. Role of aldosterone in pregnancy ............................................................................. 73
3
4.2.1 Absence of a maternal pre-eclamptic phenotype in AS-/- mice during pregnancy. 73
4.2.2 Presence of a feto-placental phenotype in AS-/- mice on control diet ................... 73
4.2.3 High salt diet does not increase intrauterine survival, but improves fetal growth in
AS-/- mice and lowered systolic blood pressure irrespective of the presence of
aldosterone .................................................................................................................. 76
4.2.4 Expression of the hypoxia inducible factor HIF1 in the placenta ........................ 78
4.2.5 Expression of pro-inflammatory factors in necrotic placentas .............................. 83
5 DISCUSSION................................................................................................................... 85
5.1 Role of aldosterone in renal potassium excretion....................................................... 85
5.1.1 Adaptation to high potassium diet is possible without aldosterone ...................... 85
5.1.2 Aldosterone independent regulation of ROMK and ENaC ................................... 86
5.1.3 Angiotensin II dependent potassium excretion .................................................... 87
5.1.4 Polyuria-dependent potassium excretion? ........................................................... 89
5.1.5 Aldosterone-independent down regulation of NCC contributes to K+ secretion .... 90
5.1.6 Potassium induced activation of Na+ and K+ channels in colon is aldosterone
dependent .................................................................................................................... 91
5.2 Role of aldosterone in pregnancy .............................................................................. 94
6 PERSPECTIVES ............................................................................................................. 98
REFERENCES ................................................................................................................... 99
ACKNOWLEDGEMENTS ................................................................................................. 109
CURRICULUM VITAE....................................................................................................... 110
4
SUMMARY
Aldosterone is a mineralocorticoid hormone with pleiotropic actions in the body.
Among others, aldosterone controls sodium and potassium excretion in kidney and
colon and it was proposed to play an important role during pregnancy. Most of the
previous studies examining the in vivo role of aldosterone have been performed in
adrenalectomized animals. However, the often incomplete removal of adrenal tissue
and the existence of local aldosterone producing systems in various organs
complicated deciphering the precise role of aldosterone. In this thesis, we took
advantage of the recently developed aldosterone-synthase-deficient (AS-/-) mice to
analyse the role of aldosterone in the control of potassium homeostasis and
pregnancy. AS-/- mice lack any endogenous aldosterone production, have slightly
elevated plasma corticosterone levels, a reduced bodyweight despite increased food
intake, but are otherwise clinically normal.
AS-/- and AS+/+ mice were kept for two days either on a standard diet (0.8%
K+), or diets enriched with potassium (2% K+, 3% K+ and 5% K+). The AS-/- mice
adapted well to 2% K+ diet. Urinary K+ excretion rose similar in AS-/- and AS+/+ mice
and plasma K+ levels remained in the normal range. With 3% K+ diet,
AS-/- mice
tended to become hyperkalemic and on 5% K + diet, the AS-/- mice were fully
decompensated and exhibited severe hyperkalemia and K +-food avoidance.
Therefore, all following studies were performed on mice kept either on standard or
on 2% K+ diets. First, we analyzed the effect of dietary K+ loading on the abundance
and subcellular localization of the renal outer medullary potassium channel (ROMK)
and the epithelial sodium channel (ENaC) in the kidney. ROMK and ENaC are
expressed in the aldosterone-sensitive distal nephron (ASDN) comprising the late
distal convoluted tubule (DCT2), the connecting tubule (CNT), and the collecting duct
(CD). ROMK is thought to mediate most of renal K+ excretion, while Na+reabsorption via ENaC provides the electrochemical driving force for K+ secretion
across the apical plasma membrane. Accordingly, we found that dietary K + loading
(2% K+) causes a translocation of ROMK and ENaC from intracellular compartments
to the apical plasma membrane of the ASDN. The apical translocation of ENaC was
accompanied by a proteolytic cleavage of the - and -subunits of ENaC, which is
thought to activate ENaC. Apical translocation of ROMK and ENaC, as well the
proteolytic activation of ENaC was similar in AS+/+ and AS-/- mice. Consistently, patch
5
clamp experiments on isolated split-open tubules revealed similar whole cell
amiloride-sensitive Na+ channel currents in the early ASDN of AS+/+ and AS-/- mice.
Likewise, on the 2% K+ diet, AS+/+ and AS-/- mice showed the same natriuretic
response to a single amiloride injection. To address the possible mechanism for the
aldosterone-independent activation of ENaC in the early ASDN, we treated AS+/+ and
AS-/- mice on 2% K+ diet with the angiotensin II AT1 receptor antagonist losartan.
While AS+/+ mice tolerated losartan treatment without any problems, AS-/- mice
decompensated and developed severe hypokalemia with K +-food avoidance.
Immunohistochemistry revealed that losartan-treatment of AS-/- mice impaired the
apical translocation of ENaC. Interestingly, AS-/- mice showed reduced abundance
and phosphorylation of the thiazide-sensitive NaCl-cotransporter (NCC), which was
present already on standard diet and became even more pronounced on high K +diet. Consistent with lowered NCC activity, AS-/- mice on 2% K+ diet showed lesser
natriuresis than AS+/+ mice in response to hydrochlorothiazide. Ussing-type chamber
experiments on isolated colon mucosa from AS+/+ and AS-/- mice showed that dietary
K+ intake (2% K+ for 2-4 days) increases Ba+-sensitive K+ channel and amiloridesensitive Na+ channel currents only in AS+/+ but not in AS-/- mice. Taken together our
results suggest that aldosterone-deficiency does not prevent the K+-intake-induced
functional adaptation of ROMK and ENaC in the kidney, but impairs the homeostatic
response of BK and ENaC in the colon. Angiotensin-II dependent activation of ENaC
and functional downregulation of NCC in the renal tubule likely contribute to the
maintenance of K+ homeostasis in the absence of aldosterone. NCC downregulation
in DCT enhances Na+ delivery to the ASDN, which can be reabsorbed via
angiotensin II activated ENaC and hence increases the driving force for K+-secretion
via ROMK.
During normal pregnancy, plasma volume expands to increase the perfusion
of the placenta and support fetal growth. The extension of plasma volume coincides
with a gradual increase in plasma aldosterone levels. Clinical studies revealed
aldosterone deficiency in a subgroup of women in pregnancy-associated disorders
like pre-eclampsia. Therefore, we aimed to test in our mouse model the hypotheses
whether chronic aldosterone deficiency may contribute or cause pre-eclampsia or
intra-uterine growth restriction (IUGR). In order to avoid differing genotypes of pups
as a confounding factor, we used only two types of breeding (AS+/+ male mating with
6
AS-/- female and AS-/- male mating with AS+/+ female) giving heterozygous pups only.
Hypertension and proteinuria are considered as cardinal signs of pre-eclampsia.
Therefore, we measured systolic blood pressure (SBP) and proteinuria in AS+/+ and
AS-/- females to check for the development of pre-eclampsia. AS-/- mice did not
develop hypertension or proteinuria but showed significantly decreased litter sizes.
To investigate the possible mechanism for decreased litter sizes, pregnant mice
were sacrificed on day 18 of pregnancy. At this stage, AS-/- females already showed
a reduced number of fetuses that correlated with an increased number of necrotic
placentas. Also, the weights of the fetuses and the placentas were lower in AS-/- than
in AS+/+ females. Histology of healthy placentas from AS+/+ and AS-/- mice showed
similar structures including a normal decidua basalis, junctional zone, and labyrinth,
while necrotic placentas showed severe coagulative necrosis and infiltration of
lymphocytes in all three layers. Consistently, necrotic placentas revealed increased
mRNA levels of the pro-inflammatory cytokine tumor necrosis factor 1alpha (TNF1),
and the monocyte chemo-attractant protein 1 (MCP-1). We hypothesized that
aldosterone deficiency decreases plasma volume and arterial perfusion, which finally
compromise placental and foetal growth due to placental hypoxia. Consistent with
placental hypoxia, we found a significantly increased protein expression of hypoxia
inducible factor 1 (HIF1 ) in placentas of AS-/- mice when compared with AS+/+ mice.
In order to improve plasma volume during pregnancy, we fed a high salt diet
(5% NaCl) to females. AS+/+ and AS-/- female mice responded well to high salt diet as
indicated by an increased SBP during high salt diet. During pregnancy, SBP
decreased in both genotypes. Litter size was slightly improved but necrotic placentas
were still observed in AS-/- mice. However, increased weight of pups was observed in
AS+/+ and AS-/- females on high salt diet during pregnancy compared to control diet.
In mice of both genotypes, placental efficiency was significantly improved by the high
salt diet suggesting a better perfusion of the placenta, which may have improved
growth of the fetuses. Accoridingly, HIF1
expression levels were similar in
placentas from AS+/+ and AS-/- females on high salt diet.
In summary, using AS-/- mice, we revealed aldosterone-dependent and
independent mechanisms for K+ homeostasis in the kidney and the colon. The
aldosterone-independent mechanisms allow for maintenance of K +-homeostasis in
the absence of any aldosterone by activation of ROMK and ENaC in the kidney and
7
perhaps down-regulation of NCC. In contrast to the kidney, the colonic adaptation to
dietary K+ intake appears to strictly depend on aldosterone. We also demonstrated,
using AS-/- mice, that aldosterone deficiency affects placental function during
pregnancy
leading
to
reduced
placental
and
fetal
growth.
Dietary
salt
supplementation during pregnancy can partially improve placental function.
8
DEUTSCHE ZUSAMMENFASSUNG DER DOKTORARBEIT
Aldosteron ist ein Mineralokortikoidhormon, das hauptsächlich in der Nebenniere
gebildet wird und unterschiedlichste Körperfunktionen beeinflussen kann. Unter
anderem spielt Aldosteron eine zentrale Rolle bei der Regulierung der Natrium- und
Kaliumausscheidung
durch
Nieren
und
Dickdarm.
Auch
während
der
Schwangerschaft scheint Aldosteron eine wichtige Funktion zu haben.
Bisherige in vivo Untersuchungen zur physiologischen Bedeutung von
Aldosteron wurden vor allem an adrenalektomierten Tieren durchgeführt. Die oft
unvollständige Entfernung der Nebennieren und eine gewisse extraadrenale
Produktion von Aldosteron erschwert
aber eine eindeutige Interpretation der
gewonnenen Daten. In dieser Doktorarbeit nutzten wir Aldosteronsynthase-defiziente
(AS-/-) Mäuse, die keine endogene Aldosteronproduktion mehr haben, um die Rolle
von Aldosteron bei der Aufrechterhaltung der Kaliumhomöostase und während der
Schwangerschaft genauer zu untersuchen. AS-/-Mäuse weisen leicht erhöhte
Plasma-Kortikosteron Werte auf, haben ein stark aktiviertes Renin-AngiotensinSystem und ein reduziertes Körpergewicht, sind aber ansonsten weitgehend
unauffällig.
Wir untersuchten die Fähigkeit von AS-/- Mäusen sich an unterschiedliche
Kaliumaufnahmen anzupassen. Dazu fütterten wir AS-/- Mäuse und Wild-typ (AS+/+)
Mäuse für zwei Tage entweder mit einer Standard Diät mit einem Kaliumgehalt von
0.8% K+ oder einer K+-angereicherten
Diät mit 2% K+,
3%K+ oder 5%K+. Wir
stellten fest, dass sich die AS-/- Mäuse gut an die 2% K+-Diät anpassen können. Wie
die AS+/+ Mäuse erhöhten sie die K+ Ausscheidung im Urin und hielten so ihre
Plasma K+-Konzentration auf einem normalen Wert. Ab einer K +-Anreicherung von
3% zeigten die AS-/- aber erste Anzeichen einer Hyperkalämie. Wurde der
Kaliumgehalt der Diät noch weiter auf 5% gesteigert dekompensierten die AS -/Mäuse vollständig. Sie zeigten eine ausgeprägte Hyperkalämie und verweigerten die
weitere Aufnahme der 5% K+-Diät. Aufgrund dieser Beobachtungen wurden alle
weiteren Untersuchungen nur noch an Mäusen, die mit der Standard Diät oder der
2% K+-Diät gefüttert wurden, durchgeführt.
Zuerst untersuchten wir den Einfluss einer erhöhten Kalium Aufnahme auf die
Menge und die subzelluläre Lokalisierung des
K+-Kanals ROMK und des
9
epithelialen Na+-Kanals ENaC, die im Aldosteron-abhängigen distalen Teil des
Nephrons, dem sogenannten ASDN, vorkommen. Im ASDN vermittelt ROMK die K +Ausscheidung über die apikale Plasmamembran. Die notwendige elektrochemische
Triebkraft für die K+-Sekretion wird durch eine ENaC-vermittelten Na+-Aufnahme
aufgebaut. Hierzu passend fanden wir nach einer erhöhten Kaliumaufnahme (2%K +)
eine Verlagerung von ROMK und ENaC aus intrazellulären Kompartimenten an die
apikale Plasmamembran entlang des ASDN. Die Verlagerung von ENaC ging mit
einer proteolytischen Spaltung und Aktivierung der alpha- und der gammaUntereinheit von ENaC einher. Die apikale Translokation von ROMK und ENaC und
die proteolytische Aktivierung von ENaC beobachten wir gleichermassen in AS+/+
und in AS-/- Mäusen. Parallel zeigten Patch-Clamp Untersuchungen an isolierten
Tubuli von AS+/+ und AS-/- Mäusen ähnliche Na+-Ströme, die mit Amilorid blockierbar
waren. Beide Genotypen zeigten auch die gleiche natriuretische Antwort auf eine
Amilorid-Injektion.
Um den möglichen Mechanismus der Aldosteron-unabhängigen Aktivierung
von ENaC aufzuklären, behandelten wir die AS +/+ und AS-/- Mäuse mit Losartan,
einem Hemmer des Angiotensin II (AT1) Rezeptors. Während die AS+/+ Mäuse diese
Behandlung
problemlos
tolerierten,
dekompensierten
die
AS-/- Mäuse.
Sie
entwickelten eine deutliche Hyperkalämie und verweigerten die K +-Diät Aufnahme.
Immunhistochemische Untersuchungen zeigten, dass bei den AS -/- Mäuse nach der
Losartan-Behandlung die apikale Translokation von ENaC nicht mehr nachweisbar
war.
Interessanterweise zeigten die die AS-/- Mäuse bereits unter StandardBedingungen eine geringere Menge und Phosphorylierung des renalen NatriumChlorid-Cotransporters (NCC) als die AS+/+ Mäuse. Die Abnahme von NCC Menge
und Phosphorylierung in den AS-/- Mäuse wurde noch deutlicher, wenn die K+Konzentration
der
Diät
erhöht
wurde.
In
Übereinstimmung
mit
diesen
Beobachtungen zeigten AS-/- Mäuse auf der 2% K+-Diät eine geringere natriuretische
Antwort auf eine Thiazid-Injektion als die entsprechenden AS+/+ Mäuse.
Ussing-Kammer-Untersuchungen am Colon zeigten, dass eine erhöhte K +Aufnahme (2% K+) für 2-4 Tage sowohl Ba+-hemmbare K+-Kanäle als auch Amilorid-
10
hemmbare Na+-Kanäle aktiviert. Diese Aktivierung der K+ und Na+ Kanäle war aber
nur in den AS+/+ und nicht in AS-/- Mäusen nachweisbar.
Zusammengefasst deuten unsere Resultate darauf hin, dass das Fehlen von
Aldosteron die funktionelle Anpassunge der Niere an eine erhöhte K +-Aufnahme
nicht verhindert. Im Gegensatz dazu scheint die homeostatische Anpassung des
Colons an eine erhöhte K+-Aufnahme an Aldosteron gebunden zu sein. In der Niere
scheint
eine
Angiotensin
II-abhängige
Aktivierung
von
ENaC
und
eine
Herunteregulierung von NCC an der Aldosteron-unabhängigen Erhaltung K+Homöostase beteiligt zu sein. Die Herunterregulierung von NCC im DCT führt zu
einer erhöhten Beladung des ASDNs mit Na+, welches dann via ENaC resorbiert
wird und damit die elektrochemische Triebkraft für die ROMK-abhängige K+Sekretion erhöht.
Während einer normalen Schwangerschaft vergrössert sich das mütterliche
Plasmavolumen um die Durchblutung der Plazenta zu optimieren und damit das
Wachstum des Fötus zu begünstigen. Die Ausdehnung des Plasmavolumens geht
mit einer Erhöhung der Plasma-Aldosteron-Spiegel einher. In klinischen Studien
wurde bei einzelnen werdenden Müttern mit Präeklampsie ein Aldosteron Mangel
nachgewiesen. Daher testeten wir in unserem Maus-Modell die Hypothese, dass ein
chronischer Aldosteron-Mangel die Schwangerschaft negativ beeinflusst und eine
Präeklampsie und/oder einen intrauterinen Wachstumsverzug (IUGR) zur Folge
haben kann.
Damit ausschliesslich heterozygote Jungtiere gezeugt wurden, wurden nur
männliche AS+/+
mit weiblichen AS-/- und männlich AS-/-
mit weiblichen AS+/+
verpaart. Bei den schwangeren Weibchen wurden dann während des gesamten
Schwangerschaftsverlaufes
der
systolische
Blutdruck
(SBP)
und
die
Proteinausscheidung im Urin gemessen, um zu kontrollieren, ob die Tiere die
charakterischen Zeichen einer Präeklampsie (Bluthochdruck und Proteinurie)
entwickelten. Wir konnten keine Anzeichen eines Bluthochdruckes oder einer
Proteinurie bei den Mäusen entdecken. Die AS-/- Mütter hatten aber deutlich kleinere
Würfe als die AS+/+ Mütter. Das gehäufte Vorkommen nekrotischer Plazenten
deutete an, dass die reduzierte Wurfgrösse auf ein intrauterines Absterben der
Frucht zurückzuführen war. Nicht nur die Anzahl der Föten war bei AS -/- Müttern
11
geringer als bei AS+/+ Müttern, sondern auch das Gewicht der entwickelten Föten
und der dazugehörigen gesunden Plazenten war deutlich reduziert. Obwohl kleiner,
zeigten die gesunden Plazenten der Mütter beider Genotypen einen strukturell
unauffälligen histologischen Befund. Im Gegensatz dazu zeigten die nekrotischen
Plazenten eine lymphozytäre Infiltration und eine erhöhte Transkription für das
inflamatorische Zytokin Tumor-Necrosis-Factor 1 alpha (TNF1 ) und das Monocyte
Chemo-Attractant Protein1 (MCP-1). Wir nehmen an, dass der Aldosteronmangel
das Plasmavolumen der schwangeren Mäuse verringert. Damit ist die optimale
arterielle Blutversorgung der Plazenten nicht mehr gewährleistet, was zu einer
plazentaren Hypoxie und damit zu einem beeinträchtigten plazentaren und damit
fötalen Wachstum führt. Die Vermutung einer plazentaren Hypoxie in den AS -/Mütter wird durch die Beobachtung einer signifikant erhöhten Expression des
Hypoxie-Induzieren-Faktor 1 alpha (HIF1 ) unterstützt.
Um das vermeintlich zu geringe Plasmavolumen der AS -/- Mäuse während der
Schwangerschaft zu optimieren, fütterten wir die werdenden Mütter mit einer NaClangereicherten Diät (5% NaCl). Sowohl die AS+/+ als auch die AS-/- Weibchen
reagierten gut auf die Diät und zeigten vor und während der Schwangerschaft
erhöhte Blutdruckwerte. Durch die NaCl-angereicherte Diät konnte die Wurfgrösse
der AS-/- Mäuse deutlich erhöht werden. Auch das Gewicht der Jungtiere war sowohl
bei den AS+/+ Tieren als auch bei den AS-/- unter der NaCl-reichen-Diät deutlich
erhöht. Trotzdem wurden bei den AS-/- Mäusen weiterhin nekrotische Plazenten
gefunden. Um den möglichen Mechanismus der verbesserten Gewichtsentwicklung
der Jungtiere zu verstehen berechneten wir die plazentäre Effizienz welche durch
das Verhältnis von Plazentagrösse zu Jungtiergrösse bestimmt wird. Beide
Genotypen zeigten eine deutliche erhöht Effizienz der Plazenta was darauf
hindeutet, dass dem erhöhten Geburtsgewicht der Jungtiere eine verbesserte
Durchblutung der Plazenta zugrunde liegt.
Zusammenfassend, konnten wir durch unsere Untersuchungen and den AS-/Mäusen Aldosteron-abhängige und -unabhängige Mechanismen zur Erhaltung der
K+-Homöostase aufklären. Die Aldosteron-unabhängigen Mechanismen ermöglichen
es durch eine Aktivierung von ROMK und ENaC und eine Herunterregulatierung von
NCC in der Niere die K+-Homöostase auch bei fehlendem Aldosteron aufrecht zu
12
erhalten. Im Gegensatz dazu scheint die Anpassung des Colons an eine erhöhte K +Aufnahme an Aldosteron gebunden zu sein.
Die
Untersuchungen
der
AS-/-
Mäuse
zeigten
auch,
dass
ein
Aldosteronmangel in der Schwangerschaft die Funktion der Plazenta negativ
beeinflusst und in der Folge zu einer Reduktion des plazentalen und fötalen
Gewichts führt. Eine Supplementierung der Diät mit NaCl scheint die Effizienz der
Plazenta zumindest teilweise zu verbessern.
13
1 INTRODUCTION
Aldosterone
Aldosterone is the main mineralocorticoid hormone produced in the glomerulosa cell
layer of the adrenal glands. Aldosterone has pleiotropic actions in the body as shown
in figure 1. One of its main functions is to participate to the hormonal control of ion
and water homeostasis. Aldosterone stimulates sodium (Na+) reabsorption and
potassium (K+) secretion by the kidney. As Na+ is the primary active osmotic particle
in the extracellular space and retains water, aldosterone finally determines the
volume of extracellular fluid and hence blood pressure [1]. Aldosterone has also
effects on many other organ systems including the cardiovascular system and
metabolic organs. Moreover, local synthesis of aldosterone has been demonstrated
in tissues such as those of the heart, blood vessels and brain [2-4]. The functions of
aldosterone in these non-epithelial tissues are still a matter of debate. Recently
aldosterone has been shown to play a role during pregnancy, in which it may
contribute to plasma volume expansion, placental perfusion and hence improved
placental and fetal growth [5, 6]. In our projects, we focussed on the role of
aldosterone in K+ homeostasis and in pregnancy. As aldosterone is produced by the
enzyme aldosterone synthase (AS), AS knock out (AS-/-) mice were used to study
the role of aldosterone
Aldosterone
Extension of plasma volume
Placental growth
Water and salt homeostasis
Adipocyte differentiation
Thermoregulation
Salt and water appetite
Central BP regulation
Cardiac structure
and function
Vascular function
Figure 1. Pleiotropic role of aldosterone (adapted and modified from [7])
14
Aldosterone synthase (Cyp11b2)
Aldosterone is synthesized from cholesterol by a series of enzymatic reactions of
which aldosterone synthase (AS, Cyp11b2) mediate the last two reactions using 11deoxycorticosterone as a substrate (figure 2). Aldosterone synthase produces
aldosterone by catalyzing first the hydroxylation of 11-deoxycorticosterone to
corticosterone,
then
the
18-hydroxylation
of
corticosterone
to
18-
hydroxycorticosterone, and finally the oxidation of 18-hydroxycorticosterone to
aldosterone [8] (figure 2). Aldosterone-deficient mice were generated by the Oliver
Smithies group by targeted disruption of Cyp11b2 gene and kindly provided to us. In
these aldosterone-synthase knockout (AS-/-) mice, the first two exons of the gene
were replaced via homologous recombination with a nucleotide sequence coding for
the expression of the enhanced green fluorescent protein (EGFP). AS-/- mice lack
any endogenous aldosterone, have a reduced body weight (75% of the wild type
mice), are hypotensive and show very high levels of plasma renin and angiotensin II,
and slightly elevated levels of plasma corticosterone [9].
Figure 2. Flow chart showing function of aldosterone synthase enzyme in aldosterone
production
15
1.1 Project 1. Role of aldosterone in renal potassium excretion
1.1.1 Aldosterone synthesis and regulation
The major role of aldosterone is to stimulate sodium (Na+) reabsorption and
potassium (K+) secretion in target tissues. The zona glomerulosa cells from the
adrenal cortex contain the aldosterone synthase enzyme involved in the final step of
aldosterone production from deoxycorticosterone. The gene encoding for the
aldosterone synthase is Cyp11b2.
There are three main factors that control aldosterone production, namely angiotensin
II, plasma K+ levels, and adrenocorticotropic hormone (ACTH). Angiotensin II is
formed during extracellular volume depletion via an activation of the reninangiotensin-system. In response to a decrease in the circulating blood volume renin
is released from the granular cells in the renal juxtraglomerular apparatus. Renin
converts angiotensinogen (produced mostly by the liver) into angiotensin I by
proteolytic cleavage.Angiotensin I in turn is then converted by the angiotensin
converting enzyme (ACE) into the physiologically active angiotensin II. Angiotensin II
binds then to the AT1 receptor on the glomerulosa cells of the adrenal cortex to
stimulate aldosterone production. An increase in extracellular K+ concentrations
directly stimulates aldosterone production in the glomerulosa cells by not yet well
understood mechanism. ACTH is released from the anterior pituitary mainly to
stimulate glucocorticoid productions. However, it does also promote aldosterone
secretion although this effect is very weak [1].
1.1.2 Target tissues and specificity for the receptors
Aldosterone mediates its effect by binding to the type 1 corticosteroid or the
mineralocorticoid receptor (MR). The MR is a ligand activated transcription factor
and is a member of the steroid/thyroid hormone nuclear receptor superfamily of
ligand inducible transcription factors [10]. The MR receptor is expressed in the
epithelium lining the tubules in the distal nephron of the kidney, the distal colon, and
the ducts of salivary and sweats glands. Glucocorticoids exert their effects through
the glucocorticoid receptors (GR) [11]. Steroid binding studies showed that both,
aldosterone and glucocorticoid, have similar high affinities for the MR [10]. Plasma
levels of glucocorticoids hormones are 100 to 1000 times higher than that of
16
aldosterone (0.1 to 1nM) and theoretically expected to occupy MR. However, in
aldosterone target tissues the MR is protected by the enzyme 11 -hydroxysteroid
dehydrogenase type β (11 -HSD2) which converts cortisol and corticosterone, but
not aldosterone, to their in-active 11-keto analog. These analog are unable to
activate the MR [12]. Thus, in aldosterone target tissues specificity for the MR is
mediated by the 11 -HSD2 enzyme.
Since the eighties of the last century, several additional target tissues for
aldosterone have been defined. Expression of MR was reported in a number of nonepithelial tissues including the pituitary, neurons of the central nervous system
(CNS), cardiac myocytes, and endothelial and smooth muscle cells of the large
vessels [10-13]. Cardiac myocytes [14], vascular cells [15] and certain areas of the
adult brain, related to control of blood pressure and salt appetite, appear to express
11 -HSD2 [16] suggesting that aldosterone may exert specific effects at this sites,
despite the high circulating glucocorticoid levels. Nevertheless, the physiological role
of the MR or aldosterone in these non-epithelial tissues has remained largely
unknown whereas patho-physiological effects of aldosterone at these sites are the
subject of intense studies [17]. Aldosterone was shown to contribute to cardiac
fibrosis and cardiovascular remodelling in animals on high salt diet [18]. There is also
evidence that aldosterone contributes to inflammation, fibrosis and progression of
cardiovascular diseases, including hypertension and metabolic syndrome [19].
Interestingly, expression of the 11 -HSD2 and the MR in human placenta was also
reported indicating a role of aldosterone in pregnancy [20]. The possible role of
aldosterone in pregnancy will be discussed in more detail in a later part of the
introduction.
1.1.3 Potassium homeostasis - External and internal K+ balance
Control of K+ homeostasis is vital for the function of many cells in the body. A high
intracellular concentration of K+ is important for cell growth whereas the maintenance
of extracellular K+ concentrations is important for normal functions of neurons,
cardiac myocytes and skeletal muscle [1, 21]. Hypokalemia or hyperkalemia result in
cardiac arrhythmias and dysfunction of neurons and muscle contraction (weakness
and cramps). The amount of K+ in the extracellular space is rather low. The K+
content of a standard meal could be already sufficient to increase extracellular K+
17
concentration. Thus, dietary K+ intake has to be managed by rapid redistribution of
taken-up, extracellular K+ into intracellular tissue stores, particularly in muscle and
liver cells. Eventually K+ is released back into the extracellular fluid and gets
excreted by the kidney into the urine [21]. Safeguarding of plasma K+ is achieved by
synchronised mechanisms of external and internal K+ balance. External K+ balance
involves the rate of K+ intake (approx.100 mEq/day) and the rate of K+ excretion
through urine (approx. 90 mEq/day) and faeces (approx. 10 mEq/day). The internal
K+ balance consists of distribution of K+ between extracellular fluid (approx. 70
mEq/day) and intracellular spaces in bone, liver, muscle and red blood cells (figure
3). This distribution is regulated by several hormones like insulin,
-adrenergic
agonists (e.g. epinephrine) and aldosterone all promoting the transport of K+ from
extracellular fluid to intracellular fluid compartments via the ubiquitous Na+-K+ATPase pump. This process of distribution can be further affected by acid-base
balance and the tonicity of the plasma [1, 22].
Figure 3.
Potassium homeostasis. External and internal K+ balances (adapted and
modified from [22]). Detail description in text.
18
External K+ balance is mainly determined by two organs, namely the kidney
and the colon. The kidneys play a major role in K+ homeostasis by excreting 90 to
95% of the daily K+ intake while the colon excretes 5 to 10% of the daily K+ intake.
Although the colon can increase K+ excretion under certain conditions like in
response to adrenal hormones, decreased renal excretory capacity and high K+ diet,
the colon alone cannot excrete K+ to the extent to maintain normal plasma K+ levels
[1]. K+ secretion is under tight control of aldosterone, which acts as the final
endocrine signal of the renin–angiotensin-aldosterone system and targets the
epithelia in the kidney and colon [17].
1.1.4 Potassium handling in the kidney
In the kidney, potassium is freely filtered in the glomerulus, almost completely
reabsorbed in the proximal tubule (almost 80%) and thick ascending limb (TAL)
(10%), secreted in the connecting tubule (CNT) and cortical collecting duct (CCD)
and reabsorbed in the outer medullary collecting duct (OMCD).
Potassium absorption
Figure 4 depicts the different cell models for K+ absorption along the nephron.
Figure 4. Potassium absorption along the nephron. Cell models showing similarities of
transporters in the basolateral membrane and different transporters at the apical membrane,
(adapted and modified from [23])
19
In the proximal tubule, K+ is reabsorbed by two paracellular mechanisms:
solvent drag and passive diffusion. Active Na+ transport in the proximal tubule drives
net fluid absorption and drags K+ along with the fluid. The transcellular ion transport
also generates at the end of the proximal tubule a lumen-positive transepithelial
voltage that provides an electrochemical gradient for K+ absorption through the low
resistance paracellular pathway. In the TAL, K+ is mostly recycledthrough the Na+K+-2Cl- -cotransporter (NKCC2) and the ROMK channel [1, 23, 24].
1.1.5 Potassium secretion - Critical role of the ASDN
The amount of K+ that finally becomes excreted in the urine critically depends on
controlled K+ secretion along the aldosterone-sensitive distal nephron (ASDN). The
ASDN comprises the late distal convoluted tubule (DCT2), the connecting tubule
(CNT), and the collecting duct (CD) [22, 24]. In the ASDN, two morphologically
distinct cell types are present namely principal cells (PC) and intercalated cells (IC)
[22, 25] (figure 5). The PCs are involved in K+ secretion, whereas ICs are involved in
K+ absorption [26, 27]. Principal cells express epithelial Na+ channels (ENaC) and
renal outer medullary K channels (ROMK) localized to the apical membrane and the
Na+-K+-ATPase on the basolateral membrane [28]. Transepithelial sodium
absorption via ENaC and Na+-K+-ATPase is electrogenic and generates a lumennegative transepithelial potential difference that acts as a driving force for K+
secretion via apical K+ channels such as ROMK and the flow dependent maxi K+
channels (BK) [24, 29-31]. Potassium secretion in the PC is a two step process that
involves first an active uptake of K+ across the basolateral membrane by Na+-K+ATPases and then second the passive diffusion of K + through apical K+ channels
along the electrochemical gradient generated by ENaC and the Na+-K+ ATPase
(figure 4) [22]. Aldosterone stimulates K+ secretion by activation of the Na+-K+ATPase, ENaC, and perhaps also the apical K+ channels. Aldosterone sensitivity is
conferred to the ASDN by the expression of the MR and the 11 -HSD2 in the ASDN
[28, 32]. Potassium absorption by intercalated cells occurs by an active transport of
K+ via H+-K+-ATPases across the apical membrane and leaving the cell across the
basolateral membrane along a favourable K+ electrochemical gradient (figure 5) [24,
33, 34].
20
Lumen
Blood
ENaC
Na+
Na+
CNT
K+
DCT
A
ROMK
CD
MR
K+
A
K+
Aldo.
MR
Cortisol
11 HSD2
-
--
Principal cell
-
+Blood
Lumen
Na+
H+
K+
K+
K+
V-
H+
ATPase
---
H+
-
Intercalated cell
+-
Figure 5. Potassium secretion and absorption in the Aldosterone-sensitive distal
nephron. Principal cell show expression of the Epithelial Sodium channel (ENaC), the Renal
outer medullary potassium channel (ROMK), Na+-K+-ATPase, and the mineralcorticoid
receptor (MR). Intercalated cell shows expression of H+-K+-ATPase, vacuolar H+-ATPase
and Na+-K+- ATPase.
In addition to aldosterone, a variety of other factors including dietary K + intake,
vasopressin (antidiuretic hormone – ADH) and tubular flow rate [22, 23, 27, 35] have
been shown to stimulate either K+ secretion of absorption along the ASDN (figure 6).
21
Figure 6. Regulatory factors influencing K+ secretion or absorption in the ASDN
(adapted from [23].)
NCC
ENaC
Na-K-ATPase
MR
11 -HSD2
ROMK / BK
Figure 7. Expression and distribution patterns of Na+ and K+ transport systems along
the ASDN. Distribution of the MR and 11 -HSD2 and the DCT specific thiazide sensitive
NCC are also indicated. (Adapted and modified from [28]).
22
1.1.6 Structure, function and regulation of sodium and potassium
channels and transporter in the ASDN
1.1.6.1 The epithelial sodium channel (ENaC)
ENaC plays an important role in whole body Na+, K+ and fluid homeostasis.
Figure 8. Structure of the ENaC channel (as predicted by Jasti et al [36]). Adapted from
[37]
This is emphasised by the observations that gain of function mutations of
ENaC such as in Liddle’s syndrome lead to severe arterial hypertension and
hypokalemia
whereas
loss
of
function
mutations
of
ENaC
such
as
in
pseudohypoaldosteronism type 1 (PHA-I) lead to a renal salt wasting syndrome and
hypotension associated with hyperkalemia [38, 39]. ENaC is a heteromultimeric
channel consisting of three different homologous subunits ,
and
(figure 8). The
three subunits share 30 to 40% homology at the level of amino acid sequence [28,
40]. ENaC might be a heterotrimer as suggested by the crystal structure of related
acid sensing ion channel 1 (ASIC1) [36, 41]. Each subunit of ENaC consists of two
transmembrane domains (M1 and M2), a large extracellular loop and short intracellular amino and carboxy termini. It is thought that ENaC forms its pore with their
23
M2 domains [38]. Co-expression of all three ENaC subunits is necessary for full
activity of the channel. Na+ movement across the channel corresponds to the
electrophysiologically measurable ion currents and can be specifically blocked by the
diuretics amiloride and triamterene [38, 42].
ENaC is regulated by a variety of extrinsic and intrinsic factors. Figure 9 and
table 1 shows some of these factors.
Extrinsic factors
Hormonal regulation - ENaC is known to be regulated by volume regulatory
hormones such as aldosterone, arginine vasopressin (AVP), the atrial natriuretic
peptide (ANP), and other hormones such as insulin and endothelin [37]. The
hormones regulate ENaC via receptor mediated modulation of intracellular signalling
pathways including kinase cascades. Aldosterone regulates ENaC - by stimulation of
the serum and glucocorticoid-regulated kinase (Sgk1) or inhibition of the extracellular
signal-regulated kinase (ERK) [43, 44]. AVP and ANP, insulin and endothelin
regulate ENaC via protein kinase A (PKA), phosphatidylinositol 3-kinase-dependent
signalling and SRC family kinases, respectively [45-49].
Table 1. Factors regulating ENaC
Extrinsic factors
Intrinsic factors
Hormonal regulation
ENaC traffiking
Mechanosensation
Phosphorylation
Proteolytic cleavage
Na+ self inhibition and feedback inhibition
Metabolic depletion and pH
Mechanosensation - In non-epithelial tissues, ENaCs subunits were shown to be
involved in mechanosensation. The
and
subunits may play an important role in
the mechanosensitivity of neurons innervating the aortic arch and vascular smooth
muscles [50, 51]. Also in isolated rabbit CCD, ENaC might become activated by
laminar shear stress due to an increased tubular flow rate [52]. ENaC activation via
shear forces occurs likely directly via an increased opening probability (Po) of the
channel [37, 53-55].
24
Figure 9. Summary of extrinsic and intrinsic factors regulating ENaC channel (adapted
from [37]).
Proteolytic cleavage - Recent evidence indicates activation of ENaCs by proteolytic
processing by intra and extracellular proteases. One such ENaC activation protease
is furin. Furin is a serine protease, which is highly abundant in the trans-golgi
network and likely cleaves ENaC already during the biosynthetic pathway. Furin
likely cleaves the
- and the -ENaC subunits at two and one extracellular sites,
respectively [56]. The furin-dependent cleavage of the -ENaC subunit releases an
inhibitory peptide from the extracellular loop of ENaC and thereby activates the
channel [57, 58]. Another ENaC-activation protease is prostasin, which is also called
"channel activating peptidase 1 (CAP-1)". Prostasin is an extracellular protease that
likely cleaves -ENaC in the extracellular loop in close proximity to the furin cleavage
site. The combined actions of prostasin and furin release then an inhibitory peptide
from -ENaC to fully activate ENaC [59].
Intrinsic factors
ENaC trafficking - ENaC activity is also regulated by trafficking to and from the
plasma membrane. The three different ENaC subunits are synthesized in the
endoplasmic reticulum (ER) and modified along the biosynthetic pathway before they
traffic from intracellular pools to the apical plasma membrane [60]. ENaC is
endocytosed into clathrin-coated vesicles and may be recycled back to the apical
plasma membrane or is shuttled to lysosomes or proteasomes for degradation [61,
62]. Several cellular and molecular mechanisms are involved in ENaC trafficking.
One of the best studied regulatory proteins is the ubiquitin-protein ligase Nedd4-2.
25
Nedd4-2 interacts with a proline-rich PPXY (PY) motif of ENaC subunits, promotes
ubiquitylation, which is followed by endocytosis and proteasomal degradation of the
channel [63]. Another ENaC trafficking associated protein is AS160, a Rab protein
regulator, which stabilises ENaC in an intracellular compartment preventing the
channel from trafficking to the apical membrane [64].
Phosphorylation - Regulation of ENaC by kinases via direct phosphorylation of its
subunits or phosphorylation of proteins interacting and regulating the channel is well
known [28, 42]. One of the best studied kinases is the aldosterone induced kinase
Sgk1 which stimulates cell surface activity and density of ENaC via direct
phosphorylation of the -subunit [65] and indirectly via phosphorylation of Nedd4-2
and AS160 [64, 66]. Phosphorylated Nedd4-2 or AS160 are recognised and blocked
by 14-3-3 proteins, which prevents Nedd4-2-dependent endocytosis and AS160mediated intracellular retention that eventually increases the channel density and
activity at the apical plasma membrane [28, 64].
1.1.6.2 ROMK
The channel (Kir1.1) is a member of the inwardly rectifying K+ channel family [67]
with high K+ selectivity [68].
Figure 10. A schematic structural model of ROMK. A. Proposed subunit structure. B.
Two of the 4 subunits illustrate the pore helices in the M1 and M2 linker (taken from [30].
26
ROMK is a pore forming protein expressed in the apical membrane of the TAL
and PC of the ASDN [30]. X-ray crystallography of homologous K+ channels from
Streptomyces lividans [69], suggest that the ROMK channel is a tetrameric channel
complex. Each monomer consists of two membrane spanning segments (M1 and
M2) and cytoplasmic N- and C-termini with high homology to the pore-forming H5
segment of voltage-gated K+ channels [24]. Studies on the packing structure of the
M1 and M2 domains by Minor et al. suggest that the M2 segments line the pore and
are surrounded by M1 segments which also participate in subunit-subunit
interactions [70] (figure 10).ROMK channels are sensitive to intracellular pH.
Channel activity is completely inhibited by a decrease in the cytosolic pH from 7.4 to
7.0 [71, 72]. An important property of ROMK channels is the sensitivity to
intracellular ATP concentrations. High concentrations of ATP inhibit channel activity
(milimolar range) whereas low concentrations of ATP (submilimolar range) are found
to activate the channel [22, 73]. ROMK channels are also known to be regulated by
phosphorylation. The ROMK channel has three putative PKA phosphorylation sites
and stimulation of these sites increases channel activity [74, 75] either by insertion of
ROMK channels into the plasma membrane [76, 77] or by augmenting the effect of
phosphatidylinositol phosphates (PIP2) [78], which activates ROMK channels [79].
Moreover, Sgk1 stimulates ROMK channel activity by phosphorylation of a serine
residue at the N-terminus of ROMK [80]. Protein kinase C (PKC) has both
stimulatory as well as inhibitory effects on ROMK channels as phosphorylation
induces export of channels to the cell membrane but decreases the sensitivity of
channels to PIP2 [81, 82]. The ROMK1 channel variant is also regulated by PTK
which increases endocytosis of channels in the CCD by tyrosine phosphorylation
[24].
1.1.6.3 BK or Maxi K channels
A large conductance Ca+ activated K+ channel (also called BK or Maxi K) is reported
to be expressed in various nephron segments such as the medullary and cortical
TAL, DCT, CNT, CCD and medullary collecting duct. The functional BK channel
consist of a pore forming
subunit (Slo 1) with six transmembrane segments and
one of the four accessory
subunits ( 1, β, γ, and 4). The 1 subunit is reported
to be expressed exclusively in the CNT mostly in the PCs in mouse whereas other
27
subunits β- 4 are thought to be expressed in the CCD. BK β and γ are found to
be expressed in either IC or PC of the CCD and 4 is expressed in IC of the CNT
and CCD [24, 83]. It has been shown that BK channels are also sensitive to cellular
pH and ATP at physiological Ca2+ levels [84, 85]. BK channels may have a role in
flow induced K+ secretion along CNT and the CCD [86, 87]. This function of BK
channels was further emphasised by the failure of increasing flow induced K+
secretion in BK- subunit knockout mice. Interestingly, net K+ excretion in response
to high K+ diet was normal in the knockout mice indicating that K+-diet induced
ROMK channels may compensate for non-functional BK channels [88]. Similarly, BK
channels appear to contribute to urinary K+ excretion in ROMK-deficient mice,
suggesting that ROMK and BK channels are somehow redundant in function and
can partially compensate for each other [89].
1.1.6.4 NCC
As shown in figure 7, the ASDN begins within the distal convoluted tubule (DCT). In
the DCT, sodium transport depends on the thiazide-sensitive sodium-chloride cotransporter (NCC), which is co-expressed with ENaC in the late DCT (DCT2).
Figure 11. Phosphorylation and activation of NCC (adapted and modified from [91] )
28
NCC, encoded by the Slc12a3 gene, is a member of the cation-chloride
cotransporter family which also includes the Na+-K+-2Cl- cortransporters NKCC2 and
NKCC1 [90]. In the distal tubule NCC plays a critical role in NaCl reabsorption and,
indirectly in, K+ homeostasis [91].A recent study has shown a decrease in the
amount of total and surface NCC due to dietary K + intake indicating a role of NCC in
K+ homeostasis [92] NCC and two other members of the family, NKCC2 and
NKCC1, are known to be activated by phosphorylation at their amino termini in
response to low chloride concentrations. Recently it has been shown that
mammalian sterile 20 (STE20)-like kinases STE20/SPS1-related proline/alanine-rich
kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1) phosphorylate NCC
at three specific residues: Thr46, Thr55 and Thr60 in human NCC. The regulation of
NCC by complex interactions of different kinases is shown in figure 11. The SPAK
and OSR1 enzymes are further phosphorylated and activated by another set of
kinases, With-No-Lysine Kinase 1 and 4 (WNK1 and WNK4). Recently, it has been
shown that WNK3 also stimulates NCC activity by inhibiting WNK4.
All WNK kinases 1, 2 and 3 are expressed in the DCT. A shorter transcript of
WNK1 lacking its kinase domain is expressed only in the kidney (Ks-WNK1)
stimulates NCC activity by blocking the inhibitory effect of WNK-1L on WNK4.
Aldosterone has been shown to activate NCC through Sgk1 dependent
phosphorylation of WNK4 kinase thus possibly blocking the inhibitory effect of WNK4
on NCC [91, 93].
The critical role of NCC for Na+ and K+ homeostasis is evidenced by genetic
diseases that over-activate or block NCC function. In Familial Hyperkalemic
Hypertension, also known as Gordon’s syndrome or Pseudohypoaldosteronism type
II (PHAII), mutations in WNK kinases causes an abnormal high activity of NCC
leading to hypertension, hyperkalemia and metabolic acidosis [94]. In contrast, lossof-function mutations within NCC cause Gitelman-syndrome characterized by renal
Na+ wasting, hypokalemia and metabolic alkalosis [95]. Interestingly, some of the
patients with Gitelman-syndrome have mutations at position 60 confirming the critical
relevance of the serine residue at this site for the activation of NCC via
phosphorylation [96].
29
1.1.7 Potassium handling by the colon
The colon does also participate in the control of K+ homeostasis. Although under
standard conditions, only 10% of the ingested K+ is excreted via the colon, colonic K+
excretion may dramatically rise and become crucial in chronic renal insufficiency
[97]. On the other hand, intestinal K+ loss during diarrhoea can lead to extreme forms
of acute hypokalemia [98]. The colon can participate in active K+ absorption and
secretion [99, 100]. Under normal conditions, the proximal colon secretes K+ while
the distal colon contributes to net K+ absorption via the non-gastric H+/K+-ATPase
(HK β) present in the apical membrane of the surface cells [97]. On a high K diet,
both proximal and distal colon secretes K+ via trans- and paracellular pathways [101,
102].
Lumen
Blood
ENaC
Na+
Na+
K+
K+
BK
Na+
K+
K+
-
--
Colonocyte
-
+-
Figure 12. Colonocyte: showing different channels and transporters in the apical and
basolateral membranes.
The transcellular pathway involves active transport of K+ into the cell via the
basolateral Na+/K+ pump and the co-transporter NKCC1. K+ is then secreted through
apical K+ channels [103, 104] that is in the colon the BK channel [97, 105]. In
addition, to transcellular K+ secretion, paracellular K+ fluxes have been described
[103]. Both the transcellular and the paracellular pathway for net K+ secretion
depend on the lumen negative transepithelial voltage [106, 107], which is like in the
kidney generated by transepithelial sodium reabsorption via ENaC (figure 12) [97].
Moreover, like the ASDN, the distal colon is aldosterone-sensitive and aldosterone
plays an important role in the regulation of K+ transport in the colon as well [108].
30
1.2 Project 2. Role of aldosterone in pregnancy
1.2.1 Pregnancy specific disorder- Pre-eclampsia
Pregnancy is characterized by a profound volume expansion due to high aldosterone
levels meant to support fetal well-being [6]. Pre-eclampsia is a clinical syndrome in
pregnant women defined as the new onset of hypertension and proteinuria after 20
weeks of gestation. It affects 3-5% of all pregnancies and is the leading cause of
maternal and fetal morbidity and mortality. The criteria to define hypertension are a
systolic blood pressure level ≥140 mmHg or a diastolic level ≥90 mmHg on two or
more occasions at least 4 to 6 hr apart. Proteinuria is defined as the urinary
excretion of more than 0.3 g protein in 24 hr urine samples, or a protein
concentration of more than 0.3 g/l protein or ≥1+ on dipstick test strips when two
random urine samples are taken 4-6 hr apart. The etiology of pre-eclampsia is
unknown but is likely to be multifactorial [109]. Risk factors include a previous history
of pre-eclampsia, multiple pregnancies, nulliparity, high body mass index before
pregnancy, maternal age older than 40 years, very young maternal age, diabetes
mellitus, renal disease, chronic hypertension, metabolic syndrome, hypercoagulable
states, and more than 10 years since the last pregnancy [109, 110].
1.2.2 Pregnancy specific disorder- IUGR
Another pregnancy specific disorder is intrauterine growth restriction or retardation
(IUGR). IUGR is the impairment of fetal growth due to anatomical and/or functional
disorders and diseases of the feto-placental-maternal unit [111]. IUGR is usually
defined as less than 10% of the predicted fetal weight for gestational age and, if not
diagnosed properly, may result in significant fetal morbidity and mortality [67]. IUGR
is further subdivided into symmetrical and asymmetrical IUGR. Symmetrical IUGR
consists of low weight, length and head circumference and is usually indicative of
processes originating early in pregnancy. In contrast asymmetrical IUGR consists of
sparing of fetal head circumference and length (relatively large head with
undergrown trunk and extremities) indicative of processes occurring in the later
stages of gestation. Symmetrical IUGR is caused by several diseases including
genetic disorders (trisomies, other chromosomal disorders and constitutional), dwarf
syndromes, congenital viral infections, some inborn errors of metabolism and
intrauterine drug exposures. Asymmetrical IUGR is mostly due to impaired utero31
placental function or nutrient deficiency. There are many different risk factors for
IUGR, among them placental insufficiency and pre-eclampsia are included [112].
1.2.3 Abnormal placental implantation - common to pre-eclampsia and
IUGR
During normal pregnancy, maternal uetroplacental blood flow increases drastically to
support the growth of the fetus (figure 13). The uteroplacental arteries, also called
spiral arteries, undergo a series of pregnancy specific changes that include 1)
endovascular invasion of trophoblast and apparent replacement of endothelium and
media smooth muscle cells, 2) loss of elasticity of the arteries, 3) dilation of arteries
to wide and in-contractile tubes, 4) and finally loss of vasomotor control.
Figure 13. Modulation of spiral arteries. Comparison in non-pregnant, normal pregnancy
and pregnancy associated with IUGR or preeclampsia (adapted from [113]).
32
The remodelling of the spiral artery increases uteroplacental perfusion and
reduces maternal vascular blood flow resistance to meet the requirement of the
fetus. Moreover, vasodilation and loss of vasomotor control guarantee sufficient
blood supply to the placenta irrespective of altered regulation of vascular resistance
and blood pressure in the mother [114].
IUGR and pre-eclampsia have in common an abnormal vascular remodelling
and placental implantation. Brosens and colleagues [115] demonstrated decreased
trophoblast invasion and absence of pregnancy specific changes in spiral arteries in
pregnancies with IUGR and preeclampsia and it is now well accepted that reduced
endovascular trophoblast invasion and spiral artery remodelling are key pathogenic
features of IUGR and pre-eclampsia as shown in figure 9. Basic research and clinical
data indicate that the inadequate invasion of uteroplacental arteries results from the
concerted action of intrinsic fetal factors (abnormal biology of extravillous
trophoblasts) and extrinsic maternal uterine factors including impaired decidual
remodeling, impaired function of uterine natural killer (NK) cells and expression of
endothelial adhesion molecules [110, 113]. Hypoxia is considered to be a central
feature of both IUGR and pre-eclampsia [116-118]. Hypoxia driven dysregulation of
placental angiogenesis via hypoxia inducible factor (HIF) may be a key to the altered
trophoblast maturation associated with IUGR or pre-eclampsia. Pro-inflammatory
cytokines can influence HIF which in turn can alter the expression of cytokines and
therefore modify trophoblast invasion [118-123]. Low oxygen tension has been
demonstrated to inhibit the normally invasive phenotype of cytotrophoblasts needed
for deep placental implantation [124]. Several animal models showing pre-eclampsia
like syndromes have been constructed by disruption of uterine, placental, or renal
blood flow and indicated that prepregnancy endothelial dysfunction underlies hypoxia
induced shallow placentation [125, 126].
1.2.4 IUGR and pre-eclampsia may have different pathophysiologies
Although endothelial dysfunction appears to be a common predisposing factor for
both IUGR and pre-eclampsia, the clinical outcome and the consequences for the
mother might be completely different. Therefore some authors suggest that IUGR
and pre-eclampsia may represent pathophysiologically separate entities [127].
However, other authors suggest that depending on the presence of other co33
morbidities such as obesity, metabolic syndrome, coagulopathies, and maternal
inflammation, predisposed pregnant women may either develop IUGR only or the
full-blown phenotype of pre-eclampsia (figure 14) [118].
Figure 14. Proposed pathogenesis of IUGR and Pre-eclampsia (adapted from [118].
In fact, tobacco smoking causes marked endothelial dysfunction and cytokine
elevation [128], but it is strongly associated with IUGR only and surprisingly inversely
correlated with pre-eclampsia [129, 130]. Tobacco smokers tend to be lean and so
probably are protected from metabolic syndrome and thus from development of preeclampsia whereas the relatively rare group of smokers with concomitant metabolic
syndrome may have elevated risk for developing fulminant pre-eclampsia [118].
1.2.5 Placental efficiency- an important factor for fetal growth
Size and growth of the fetus at birth are important for determining the morbidity and
mortality immediately after birth and during later life. Children small or large for
34
gestational age or with IUGR are more prone for developing adult-onset
degenerative diseases, such as type 2 diabetes and glucose intolerance [131]. An
important factor or process for fetal growth is placental efficiency [132]. The most
simple definition of placental efficiency is the gram of fetus produced per gram of
placenta [133]. It can also be measured as grams of fetus produced per unit area of
placental exchange surface but this definition is used very rarely due to the difficulty
to measure the exchange area in every placenta [134]. Indeed, in many species, a
positive correlation has been shown between fetal weights near term to placental
weight as a proxy measurement of surface area for transport of nutrients [134, 135].
In turn, size of the placenta, morphology, blood flow and abundance of transporters
determine the nutrient transfer capacity of the placenta [136]. In addition, synthesis
and metabolism of nutrients and hormones by uteroplacental tissue influence the
rate of fetal growth [137]. Therefore, intrauterine growth can be affected by changes
in any of the above mentioned placental factors [136, 138].
1.2.6 Role of angiogenic and antiangiogenic, and vasoactive factors in
the development of pre-eclampsia
The improper opening of the spiral arteries with subsequent placental hypoxia is
thought to trigger the release of vasoactive factors from the placenta into the
maternal circulation that finally causes systemic endothelial dysfunction and the full
blown syndrome of pre-eclampsia (figure 15). Among other factors, soluble fms like
tyrosine kinase 1(sFlt1) [139-141] appears to play a crucial role in the pathogenesis
of pre-eclampsia. The Fms like tyrosine kinase receptor (Flt1) is highly expressed in
the developing placenta and mediates the angiogenic effects of the vascular
endothelial growth factor (VEGF) and the placental growth factor (PlGF) during
invasion of the trophoblasts. Most of the Flt produced in the placenta during the later
stages of normal gestation is a soluble form (sFlt), which is generated by alternative
splicing of the Flt mRNA. The sFlt lacks the transmembrane and cytoplasmic
domains of Flt1 and is released in large amounts into blood where it reduces the
level of free VEGF and PlGF via soluble antagonism. In pre-eclampsia, sFlt is
produced in large amounts already during early pregnancy.
35
Figure 15. Role of the antiangiogenic factor sFlt 1 in the maternal syndrome of preeclampsia (adapted from [110]).
This premature production of sFlt leads to an inappropriate decrease of VEGF
and PlGF levels in the maternal circulation causing endothelial dysfunction of
maternal vessels and the maternal pre-eclamptic syndrome [110]. Consecutively,
blood pressure (BP) increases and the pre-eclamptic phenotype will develop
damaging maternal organs such as kidneys, liver and brain [109, 110, 139-141].
In addition to VEGF signaling, also transforming growth factor (TGF) signaling
appears to be affected in the vasculature of pre-eclamptic women. Clinical studies
demonstrated that the soluble Endoglin (Eng) is elevated in the sera of pre-eclamptic
individuals. Membrane bound Eng is a cell-surface coreceptor for transforming
growth factor (TGF)-
1 and TGF-
γ that modulate the actions of TGF- 1 and
TGF- γ in endothelial cells and the syncytiotrophoblast. Soluble Eng neutralizes
circulating TGF-
1 before binding to its receptors and hence impairs TGF-
signaling in the vasculature [141] leading to activation of eNOS and vasodilation.
36
Figure 16. Hypothesis of pre-eclampsia pathophysiology (adapted from [110])
Arterial hypertension in pre-eclampsia might be also explained, at least in
part, by the observed increased plasma concentrations of vasoconstrictory
substances (e.g. endothelin, thromboxane) and decreased plasma concentrations of
vasodilatory mediators (e.g. prostacyclin, NO) in pre-eclamptic women [110]. Several
lines of evidence have implicated the role of the renin–angiotensin system in the
pathogenesis
of
pre-eclampsia.
During
normal
pregnancy,
plasma
renin
concentration, renin activity and angiotensin II levels are increased. However,
pregnant women remain normotensive which is likely related to a reduced vascular
responsiveness to angiotensin II [142]. In pre-eclampsia, the levels of plasma renin
activity, the concentration of angiotensin II and aldosterone are decreased, but the
pre-eclamptic patients manifest an exaggerated pressor response to angiotensin II
[142, 143].
Thus, the pathophysiology of proteinuria and hypertension in pre-eclampsia
may involve the imbalanced expression of a variety of vasoactive substances
37
including VEGF, prostaglandins, thromboxane and angiotensin II. The schematic
figure 16 summaries a unifying hypothesis of pre-eclampsia pathophysiology.
1.2.7 Possible role of aldosterone
pre-eclamapsia
in the development of the
Normal pregnancy is characterized by a marked expansion of plasma and
extracellular volume associated with changes in renal hemodynamics as well as in
the circulating level of adrenal steroid hormones [144, 145], which likely help to adapt
the maternal circulation to the increasing blood and substrate requirements of the
growing placenta and fetus. Consistently, plasma aldosterone concentrations
increase during normal pregnancy coinciding with plasma volume expansion [146,
147]. In pregnancies later destined for pre-eclampsia, a reduced plasma volume
precedes the onset of the disease [147-149] while plasma aldosterone concentration
and renin activity are paradoxically suppressed [150]. This observation suggested
pre-eclampsia to be caused or aggravated by an inadequate balance of extracellular
volume and the activity of the renin-angiotensin-aldosterone system. As a substantial
group of pre-eclamptic women show reduced aldosterone synthase enzyme
(Cyp11b2 gene) activity [151], aldosterone deficiency was implicated to play an
important role in the pathogenesis of pre-eclampsia. Aldosterone deficiency may
lower extracellular volume and increase thereby the risk of placental hypoperfusion
[151] with subsequent release of vasoactive factors leading to pre-eclampsia. The
possible role of aldosterone was further evidenced by the finding that loss- and gainof function due to distinct gene variants of the Cyp11b2 may predispose for and
protect from pre-eclampsia, respectively. In fact, the V386A variant of Cyp11b2
gene, which causes a deficiency in the rate-limiting step of aldosterone synthesis,
namely the 18-hydroxycorticosterone methyl oxidase activity [152], was observed
solely in a subgroup of pre-eclamptic women, but never in normal pregnant women
[151]. In contrast, the gain of function variants of the Cyp11b2 gene, SF-1 and Int2
(C), which have been shown to be associated with hypertension in non-pregnant
women
[153], apparently reduce the risk of developing pre-eclampsia [154].
Recently, observations by Gennari-Moser et al. [5] suggest a role of aldosterone for
fetal perfusion and also for placental growth. Taken together, these studies propose
a role of aldosterone availability for placental size, fetal perfusion and also the
development of pre-eclampsia.
38
1.2.8 Different components
expressed in placenta
of
aldosterone
effector
mechanism
The following figures 17 gives an overview on the different placental cell types
involved in the development of the placenta.
Figure 17. Development of the placenta from the embryonic day E 3.5 to E 12.5
(adapted from [155]).
The syncytiotrophoblast acts as a transporting barrier that regulates the
transfer of nutrients, solutes and water between maternal and fetal blood. In the
human trophoblast, expression of the mineralocorticoid receptor (MR) and 11 HSD2
mRNA
have
been
detected
[156].
In
addition,
MR
protein
was
immunolocalized to human syncytiotrophoblast and cytotrophoblast cells, and 11 HSD2 was found in the syncytiotrophoblast [20]. Driver et al. [157] demonstrated
that primary cytotrophoblast cell cultures express the Na+/K+-ATPase ( 1 and
1
subunits), - and -subunits of ENaC and the Sgk1. Sgk1 was strongly and rapidly
induced by corticosteroids (aldosterone and dexamethasone). Aldosterone induced
Sgk1 activation was not inhibited by a GR antagonist indicating the presence of
functional MR receptors in the placental trophoblast cells. Placental 11 -HSD2 may
protect the MR in a fashion analogous to classical mineralocorticoid target tissues to
modulate trophoblast sodium transport [156].
Significantly decreased 11 -HSD2
mRNA expression levels were observed in placentas of patients with pre-eclampsia
[158].
In syncytiotrophoblasts from normal placenta, ENaC was present on the
apical membrane but not detected in pre-eclamptic placenta on mRNA and protein
39
level. In the BeWo cell line, a model of human syncytiotrophoblasts, ENaC was
found and its expression is regulated by aldosterone, vasopressin, progesterone and
estradiol [159]. Some evidence exists supporting the hypothesis that ENaC channels
are required for the migration of BeWo cells [160]. Recently the
role of ENaC
proteins in the migration of vascular smooth muscle cells (VSMC) has been
demonstrated in cell culture [161]. In sheep low aldosterone levels resulted in
reduced placental weight, adverse fetal outcome and up-regulation of
VEGF in
placenta [162]. VEGF stimulates aldosterone production in cultured adrenal and
endothelial cells (unpublished data from Markus Mohaupt). In vitro studies suggest
that aldosterone stimulates trophoblast proliferation [5]. These experiments suggest
that VEGF stimulates aldosterone production via the adrenal gland and aldosterone
may have a role in trophoblast proliferation/migration and in placental development.
1.2.9 High salt diet during pregnancy may affect blood pressure and
pregnancy outcome
Aldosterone is closely linked to salt reabsorption. Of interest, an old clinical study
forwards the observation of a reduced number of pre-eclampsia and fetal death on a
high salt diet [163]. In addition, in a hypertensive woman homozygous for the V386A
mutant of Cyp11b2 and low aldosterone availability, salt supplementation lowered
BP throughout pregnancy [164]. In contrast, in pre-eclampsia, the benefits of salt
supplementation are still controversial and a recent meta-analysis did not show any
beneficial impact [165].
40
2 AIMS OF THE STUDY
2.1 Role of aldosterone in renal potassium excretion
Previous observation by Muto et al. [166] on isolated perfused CCD of
adrenalectomized rabbits suggested an aldosterone-independent mechanism of K+
secretion on high K+ diet.
In this study, increased apical Na+ and K+ channels
currents were observed on high K+ diet in adrenalectomized rabbits. Another studies
in adrenalectomized rats on high K+ diet also showed an increased number of
conducting Na+ channels suggesting that there are factors other than aldosterone
that regulate ROMK-like SK channel, Na+ channels and Na+/K+ pumps on high K diet
[167]. Moreover, the presence of a local renal aldosterone system and its regulation
by salt has been suggested [168, 169]. Obviously the presence of a local renal
aldosterone system was not considered in previous studies. Therefore, to further
address the role of aldosterone for the renal and colonic control of K+ homeostasis,
we used the aldosterone synthase knockout (AS-/-) mice as an in vivo model without
any endogenous aldosterone production.
2.2 Role of aldosterone in pregnancy
Available literature suggests that aldosterone plays an important role in pregnancy.
However, it is not clear whether aldosterone deficiency alone can lead to
compromised pregnancy, IUGR, or preeclampsia and whether aldosterone
deficiency can be counterbalanced by a high salt diet. To address these questions
we used aldosterone synthase deficient knockout mice (AS-/-) [9]. Monitoring of
pregnancy in AS-/- mice for systolic BP, proteinuria, and pregnancy outcome was
performed to assess the effects of aldosterone in murine pregnancy. Detailed
analysis of placentas and pups also examined the role of aldosterone in the
development of IUGR and placental efficiency.
41
3 MATERIAL AND METHODS
3.1 Role of aldosterone in renal potassium excretion
3.1.1 Animal model
All experiments were performed on aldosterone synthase wild type (Cyp11b2, AS+/+)
and aldosterone synthase-deficient (AS-/-) mice with a inbred129Sv genetic
background [170]. This mouse model was generously provided by Prof. Oliver
Smithies, University of North Carolina, Chapel Hill, NC, USA [9]. Age-matched male
AS+/+ and AS-/- mice (8 to 14 weeks) were used for all experiments. All experiments
were performed according to Swiss Animal Welfare laws and approved by the local
veterinary authority (Veterinäramt Zürich). All animals received standard rodent chow
GLP 3433 (PROVIMI KLIBA, Kaiseraugst, Switzerland).
3.1.2 Metabolic cage experiments
To analyze adaptive physiological changes in mice, animals were kept individually in
metabolic cages (Tecniplast, Buguggiate, (VA) Italy). This type of cages allows an
exact monitoring of individual food and water consumption as well as urine and
faeces collection. Mice were adapted to metabolic cages for 3 days before the actual
measurements. During the experiment bodyweight, consumption of food and water
as well as urine volumes and faeces were monitored and collected daily. 24 hour
urine was collected under mineral oil to avoid evaporation. At the end of the
experiments urinary electrolytes (Na+, K+, Ca2+,Mg2+,Cl-,SO4-) were measured by ion
chromatography (Metrohm ion chromatograph, Herisau, Switzerland) and urine
creatinine was measured by the Jaffe method [171].
3.1.3 Experimental protocol for dietary potassium loading
Four different potassium (K+) diets were used in this study. Control diet (0.8% K;
standard powder food), 2% K+ diet (2.2 g of KCl + 97.8 g standard powder food), 3%
K+ diet (4.2 g KCl + 95.8 g standard powder food) and 5% K+ diet (8 g of KCl + 92 g
standard powder food). Powdered food with the appropriate amount of KCl was
mixed with milliQ water in 1:1 proportion (50 g food + 50 ml water) to prepare wet
diets. AS+/+ and AS-/- mice were divided into four different groups: Control group
42
(Control diet, n = 5), 2% K+ diet group (n = 7), 3% K+ diet group (n = 7) and 5% K+
diet group (n = 5). After an adaptation phase in the metabolic cage for 3 days all
mice were fed for one day with control diet (time point 0) followed by two days on the
experimental diets varying in their K+ contents. At the end of the experiment, mice
were anesthetized with ketamine/xylazine (Narketan (Vétoquinol, CH): 65 mg/kg BW;
Xylazin (Streuli, CH): 13 mg/kg BW), and heparinized venous blood from the
abdominal vein was collected and analyzed immediately for pH, blood gases, and
electrolytes on a Radiometer ABL 505 (Radiometer, Copenhagen, Denmark) blood
gas analyzer. Serum was collected and frozen until further analysis. Kidneys were
harvested, immediately frozen in liquid nitrogen, and stored at -80oC until mRNA
and/or protein extraction.
3.1.4 Changes in tubular workload
To assess the functional activity of ENaC and NCC in the kidney in vivo, AS+/+ and
AS-/- mice were treated with the diuretics amiloride and thiazide, respectively. Mice
were kept in metabolic cages and fed one day with control diet and 2 days with 2%
K+ diet to get the control physiological values. After these three days, mice were
divided into control groups and treatment group (injection of diuretics). At the end of
the experiment, the urinary bladder was emptied by massage, and mice from control
groups were treated with vehicles (0.9% saline i.p., AS+/+ n = 9, AS-/- n = 8 or
dimethyl sulfoxide (DMSO) i.p., AS+/+ n = 8, AS-/- n = 9; Control-group), and mice
from the treatment groups were injected with either amiloride (5 µg amiloride/g body
weight in 200 µl of 0.9% saline solution i.p.; Amiloride group; AS+/+ n = 11, AS-/- n =
9) or with hydrochorthiazide (50 µg hydrochlorothiazide/g body weight in 200 µl
DMSO; Thiazide group; AS+/+ n = 9, AS-/- n = 9). After 4 hours, urine was collected
from urine collector and by emptying urinary bladder by massage. Urine was
analyzed for electrolytes including Na+ and K+.
3.1.5 Role of angiotensin II in renal adaptation to high potassium diet
To assess the role of angiotensin II in compensating the lack of aldosterone, mice
were given the AT1 receptor blocker losartan. AS+/+ and AS-/- mice were individually
kept in metabolic cages. After 3 days of adaptation on control diet and one and half
days on 2 % K+ diet, mice were divided in two groups and received in the evening
43
either a s.c. injection of vehicle (milliQ water; Control group; n = 4) or losartan
(10 mg/kg body weight in 50µl milliQ; losartan group; n = 5). At the end of the
experiment, mice were anesthetized and heparinized venous blood was collected
from the abdominal vein and analyzed immediately for electrolytes. Urine was
collected under mineral oil and analyzed for creatinine, and electrolytes including
Na+ and K+.
3.1.6 Analysis of the urinary concentration capability of AS+/+ and AS-/- mice
To examine the urinary concentrating mechanism, mice were kept in metabolic
cages for 5 days. To have the same water intake in both groups, a special agar diet
was prepared that comprised the same volume of water but an amount of food that
matched the one recorded before to be eaten daily by the AS+/+ and AS-/- mice
(40 mg of agar and 4 ml of water per 1 g and 1.33 g of standard powdered food for
AS+/+ mice and AS-/- mice, respectively). After 3 days of adaptation to the 0.8% K+
agar diet, mice were given 2% K+ in the agar diet. After two days, the urinary bladder
was emptied by massage and mice were divided into two groups and injected s.c.
with either vehicle (0.9% saline; control group; n = 5) or with the modified form of
vasopressin called as desmopressin (DDAVP) (Sigma) (1 ng/g of body weight in 100
µl of 0.9% saline solution; DDAVP group; n = 5). Urine was collected before injection
and 4 hr after the treatment by emptying the urinary bladder by massage. Urine
osmolarity was measured by freezing point depression (Osmometer, Roebling). At
the end of the experiment, mice were anesthetized and heparinized venous blood
was collected and analyzed immediately on a Radiometer ABL 505 (Radiometer,
Copenhagen, Denmark) blood gas analyzer for plasma Na+ and K+ concentration.
3.1.7 RNA extraction from the kidney
Snap frozen kidneys (5 kidneys per group) were homogenized in RLT Buffer
(Qiagen) supplemented with 2-mercaptoethanol to a final concentration of 1%. Total
RNA was extracted from 200 µl aliquots of each homogenized sample using RNeasy
Mini Kit (Qiagen) according to the manufacturer’s instructions. Quality and
concentration of the isolated RNA preparations were measured by ND-1000
spectrophotometer (NanoDrop Technologies). Total RNA samples were stored at
-80oC until further use.
44
3.1.8 Quantitative Real-time PCR
Each RNA sample was diluted to 100 ng/µl and 3 µl was used as a template for
reverse transcription using the TaqMan Reverse Transcription Kit (Applied
Biosystems, Forster City, CA). Quantitative real time (qRT-PCR) was performed on
the ABIPRISM 7500 Sequence Detection System (Applied Biosystem). Primers for
the NCC (Slc12a3) were designed using online Primer 3 software. Sequences of
primers and probes are as follows: NCC forward 5’-TAG ACC CCA TCA ATG ACA
TCC-3’ and reverse 5’-AGG TAG TTG GCA AAG GAG ACC-3’ (accession number:
NM_001205311); BK 1 forward 5’-CCA GCG GAG ACC CAG AGA-γ’, reverse 5’GGG CCA TCA CCA GCT TCT T-γ’and probe 5’-CTA AAT GAC TGT TGC CTC
CAG TGG CCA-γ’ (accession number: NM_031169); BK β forward 5’-GCT GCG
CTC CTA CAT GCA-γ’, reverse 5’-GCC CAC AGC TGA AGG AAC AG-γ’ and probe
5’-AGC GTG TGG ACA GAA GAA GCC CAG TGT-γ’ (accession number:
NM_028231); BK 4 forward 5’-AAC TCC AGG GCG CTG CTA-γ’, reverse 5’-CTC
TTA CAG GGC GGG ATA TAG GA-γ’ and probe 5’-ACA GCG ACC AGC ACC AGC
TCC TG-γ’ (accession number: NM_021452). Primers for hypoxanthine guanine
phosphoribosyl transferase (HPRT) as a housekeeping gene were designed using
Primer Express software from Applied Biosystem. Primer sequences for HPRT were
forward 5’-TTA TCA GAC TGA AGA GCT ACT GTA AGA TC-γ and reverse 5’-TTA
CCA GTG TCA ATT ATA TCT TCA ACA ATC-γ’ (accession number: NM_013556)
as described previously [172]. Specificity of the primers was - tested in a standard
PCR and always resulted in a single product of the expected size on 2% agarose
gel. Real time PCR reactions were performed for NCC using the iQ SYBR Green
Supermix (Bio-rad). ROX Passive reference Dye (Bio-rad) 3.3 µl/1.25 ml of iQ SYBR
Green Supermix was added. Briefly, 3 µl cDNA, 0.8 µl of each primer (10 µM), 5.4 µl
RNase free water, 10 µl iQ SYBR Green Supermix reached 20 µl final reaction
volume. Real time PCR reactions were performed for BK 1, BK β and BK 4 using
TaqMan Universal PCR Mastermix (Applied Biosystem). Briefly, 3 µl cDNA, 0.8 µl of
each primer (25 µM), 0.4 µl of labeled probe, 6 µl of RNAse free water and 10 µl of
TaqMan Universal PCR master mix reached total volume of 20 µl of final reaction
volume. Reaction conditions were: denaturation at 95oC for 10 min followed by 40
cycles of denaturation at 95oC for 15 s and annealing/elongation at 60oC for 60 s
with auto ramp time. For PCR reactions using the iQ SYBR Green Supermix last
45
step is followed by dissociation stage (95oC for 15 s, 60oC for 15 s followed by slow
ramp to 95oC). All reactions were run in duplicate. The expression of gene of interest
was calculated in relation to HPRT. Relative expression ratios were calculated as R
= 2[Ct(HPRT/_-actin)_Ct(test gene)].
3.1.9 Membrane preparation and western blot analysis
Total membrane proteins were prepared as described previously [173]. After
measurement of the protein concentration (Bio-rad Dc Protein Assay, Bio-rad,
Hercules, CA, USA), 50 µg of crude membrane proteins were solubilized in Laemmli
sample buffer, and SDS-PAGE was performed on 8% polyacrylamide gels. For
immunoblotting, the proteins were transferred electrophoretically to nitrocellulose
transfer membrane (PROTRAN 0.2 µM, Whatman GmbH, Dassel, Germany). After
incubation with Odyssey blocking buffer, (Li-COR) for 60 min, the blots were
incubated with the respective primary antibodies diluted in Odyssey blocking buffer
overnight at 4oC.The primary antibodies used are listed in Table 1.
Table 2. Antibodies used for immunohistochemistry and western blotting
Dilution
Antibody
Host
WB
IF
Source
ROMK1
Rabbit
1:800
1:4,000
Alomone
ENAC
Rabbit
1:5000
1: 5,000
J.Loffing
ENaC
Rabbit
1:10,000
1:40,000
J.Loffing
ENaC
Rabbit
1:10,000
1:40,000
J.Loffing
NCC
Rabbit
1:8000
1:10,000
J.Loffing
pNCCT53
Rabbit
1:1000
1:15,000
R. Fenton
pNCCT58
Rabbit
1:2000
R.Fenton
pNCCs89
Rabbit
1:4000
J.Loffing
AQP2
Rabbit
1:10,000
J.Loffing
actin
Rabbit
1:10,000
Sigma
Calbindine D28k
Mouse
BK
Mouse 1:10,000
1:10,000
Swant (CH)
NeuroMab
46
After overnight incubation, the membranes were washed three times with
phosphate buffer saline containing 0.1% Tween 20, and incubated with the
fluorescent secondary antibody (goat anti-rabbit IRdye 800 1:10,000, or goat anti
mouse IRDye 680, 1:20,000, Li-COR, Nebraska, USA or from Rockland,
Gilbertsville, PA) for 1 h at room temperature. After washing the membranes three
times, the protein signal was detected by scanning the membrane with the Odyssey
Infrared Imaging System (Li-COR Biosciences). All images were analyzed using
analysis software provided with the system to calculate the protein of interest/actin
ratio.
3.1.10 Immunohistochemistry
For immunohistochemistry, AS+/+ and AS-/- mice of the different experimental groups
(n = 5 each) were anesthetized with ketamine/xylazine (Narketan (Vétoquinol, CH):
65 mg/kg BW; Xylazin (Streuli, CH): 13 mg/kg BW) and the kidneys were fixed by
intravascular perfusion via the abdominal aorta [174]. The fixation solution contained
3% paraformaldehyde (Sigma-Aldrich, USA) in 0.1 M phosphate buffer. After 5 min
of fixation kidneys were rinsed for 5 min with 0.1 M phosphate buffer. 2 mm thick
kidney slices were subsequently mounted on thin cork slices, frozen in liquid
propane and stored at -80°C until further use.
For immunohistochemistry, kidneys were cut into 4 - 5 µm thin sections in a
cryostat (Microm, Leica, CH), and mounted on chrome-alum/gelatin coated glass
slide. After 10 min blocking of unspecific binding sites by 10% normal goat serum,
sections were incubated over night with the primary antibodies diluted in 1%
PBS/BSA. Overnight incubation took place at 4°C in a humidified chamber. Antibody
dilutions are given in table 1. After repeated rinsing of the sections with PBS, binding
sites of the primary antibodies were detected with either Cy3 conjugated goat anti
rabbit antibody or FITC conjugated goat anti mouse antibody (Jackson Immuno
Research Laboratories, USA). After three times rinsing, the slides were mounted in
Glycergel (DAKO, USA) with 2% 1,4-Diazabicyclo[2.2.2.]octans (Sigma-Aldrich,
USA) to avoid fading of the signal. Sections were studies by epifluorescence (Leica
DM6000; Leica D) and analyzed by Leica AF6000 system (Leica D).
47
3.1.11 Ussing chamber experiments
Male mice kept on the respective K+ containing diets were sacrificed by cervical
dislocation. The distal part of the colon was isolated and rinsed in ice cold ringer
solution. Only the distal 1.5 cm of the intact mouse colon was then mounted in an
Ussing Chamber. The two halves of the chamber were continuously perfused by a
bubble lift system. The solutions on the two sides were symmetrical and with the
following composition (in mM): NaCl 120; NaHCO3 25; K2HPO4 1.6; KH2PO4 0.4; Cagluconate 1.3; MgCl2 1; D-glucose 5, indomethacine (5 µM). The reservoirs were
bubbled with carbogen (5% CO2 and 95% O2) and kept at 37oC by water jackets.
Initially, tetrodotoxin (TTX, 1 µM) was added to the serosal side to inhibit possible
secretory activation by the enteric nervous system or other autonomous nerve cells.
Experiments were performed in a setup where the transepithelial voltage was
clamped to “zero” (Physiological Instruments, USA). The apertures of the clamped
chambers were 0.283 cm2. After mounting the tissues and an equilibration period of
30 min, amiloride (100 µM) was added to the mucosal perfusate to obtain a value of
amiloride-sensitive short circuit current ( Isc(amil)), a measure of electrogenic ENaCmediated Na+ absorption. After additional 5 min. BaCl2 (5 mM) was added also to
the mucosal perfusate to obtain a value of Ba 2+-sensitive short circuit current
((Isc(Ba2+)), a measurement of electrogenic BK-mediated K+ secretion.
3.1.12 Statistical analysis
Results are expressed as mean ± SEM. All data were tested for significance using
unpaired and paired Student’s test where appropriate. Only values with p < 0.05
were considered as significant.
48
3.2. Role of aldosterone in pregnancy
3.2.1 Animals
All experiments were performed on inbred 129 SvEv genetic background
aldosterone synthase wild type (AS+/+) and aldosterone synthase-deficient (AS-/-)
female mice. Mice were bred in our own animal facility. All experiments were
performed according to Swiss Animal Welfare laws and approved by the local
veterinary authority (Kantonales Veterinäramt, Zürich). Unless indicated otherwise,
animals were fed standard rodent chow (3430, KLIBA NAFAG, Kaiseraugst,
Switzerland) with free access to normal tap water.
3.2.2 Study design and treatment
Age matched female AS+/+ and AS-/- mice, between 3.5 to 6.5 months old, were used
for experiments. Animals were divided into two groups, a control group and an
experimental group. In the control group, AS-/- male were mated with AS+/+ female
mice, whereas in the experimental group, AS+/+ male mice were mated with AS-/females to generate only heterozygous pups to have the same genotype for all pups.
Both groups were fed with either the standard rodent chow powder, (3433 KLIBA
NAFAG, Kaiseraugst, Switzerland) containing 0.20% Na+ and 0.36% Cl- (AS+/+ n = 9
and AS-/- mice n = 9) or with standard diet supplemented with NaCl (high salt diet,
5% NaCl) (AS+/+ n = 6-8 and AS-/- mice n = 8-10). To feed the mice with high salt
diet, 100 g of standard rodent chow powder was mixed with 5 g NaCl and then mixed
with an equal volume of deionised water to moisten the food for feeding. Male and
female mice were kept together in individual cages for breeding and female mice
were controlled daily in the early morning for the presence of copulation plugs and
the following day was considered as gestational day 1. Systolic blood pressure
(SBP) was measured in female mice using the tail cuff method (see below). In
second set of experiments mice were fed either the control diet (AS+/+ n = 8, AS-/- n =
15) or high salt diet (5% NaCl) (AS+/+ n = 6, AS-/- n = 9) and sacrificed on day 18 of
pregnancy by cervical dislocation. The number and weight of the pups and the
placentas were measured, and tissues collected for further analysis. Placentas were
collected and rapidly frozen in liquid nitrogen and stored at -80oC for RNA extraction.
Some placentas were fixed in 4% paraformaldehyde for further histological analysis.
49
3.2.3 Tail cuff blood pressure measurements
SBP was measured by a non-invasive computerized tail cuff method (BP2000,
Visitech Systems, Apex, NC, USA) as described previously [175]. Mice were
adapted for 5 days before the start of the study. Blood pressure was measured daily
between 1 pm to 5 pm. Averages of systolic BP with a standard deviation of less
than 10 mmHg were considered to be stable measurements and were used for
further analysis. SBP from female mice on control diet was measured on control
conditions 2 to 3 days before pregnancy and from day 1 of pregnancy until the end of
pregnancy up to 21 days. Mean SBP was calculated from each day’s average SBP
measurements. SBP of animals in the high salt diet experiments was measured first
on control diet, then from day 4 to 6 of high salt diet. Thereafter, female mice were
kept together with male mice for mating and SBP was measured at gestational days
5 to 7 and days 15 to 17. Mean SBP was calculated from 3 days of average SBP
measurements.
3.2.4 Urinary protein concentration measurement
Spot urines were collected from female mice during pregnancy daily by gentle
massage of the urinary bladder. Total urinary protein concentration was measured
with a standard protein assay kit (Bio-rad Dc Protein Assay, Bio-rad, Hercules, CA,
USA). Urinary creatinine concentration was measured by the Jaffe method [171].
3.2.5 RNA extraction
Snap frozen placentas (5 placentas per group) were homogenized in RLT buffer
(Qiagen, Hilden, Germany) supplemented with 2-mercaptoethanol to a final
concentration of 1%. Total RNA was extracted from 200 µl aliquots of each
homogenized sample using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to
the manufacturer’s instructions. Quality and concentration of the isolated RNA
preparations were measured on a ND-1000 spectrophotometer (Thermo Fischer
Scientific Inc., Wilmington, DE, USA). Total RNA samples were stored at -80oC until
further use.
50
3.2.6 Semi-quantitative Real-time PCR
Each RNA sample was diluted to 100 ng/µl and 3 µl was used as a template for
reverse transcription using the TaqMan Reverse Transcription Kit (Applied
Biosystems, Forster City, CA). Quantitative real time (qRT-PCR) was performed on
the ABIPRISM 7500 Sequence Detection System (Applied Biosystem, Foster City,
CA, USA). Primers for vascular endothelial growth factor (VEGF), soluble fms-like
tyrosine kinase-1 (sFlt-1), and the hypoxia inducible factor 1 (HIF1 ) were designed
using Primer 3 online software, whereas primers for hypoxanthine guanine
phosphoribosyl transferase (HPRT) as housekeeping gene were designed using
Primer Express software from Applied Biosystem. The primers and probes used from
Applied Biosystem are monocyte chemo-attractant protein 1 MCP-1 (Mm00441242)
and Tumor Necrosis Factor 1alpha TNF
(Mm99999068). Sequences of primers
designed by ourselves are as follows: VEGF forward 5’-CAG GCT GCT GTA ACG
ATG AA-γ’ and reverse 5’-GCA TTC ACA TCT GCT GTG CT-γ’ (accession number:
NM_009505.4); sFlt forward 5’-GGG AAG ACA TCC TTC GGA AGA-γ’ and
reverse 5’-TGT GGT ACA ATC ATT CCT CCT G-γ’ (accession number: D88690);
HIF1 forward 5’-ATC TCG GCG AAG CAA AGA G and reverse
5’-CTG TCT AGA
CCA CCG GCA TC-γ’ (accession number: NM_010431); HPRT forward 5’-TTA
TCA GAC TGA AGA GCT ACT GTA AGA TC-γ and reverse 5’-TTA CCA GTG TCA
ATT ATA TCT TCA ACA ATC-γ’ (accession number: NM_013556). The specificity
of the primers was first tested in a standard PCR that resulted in a single PCR
product of the expected size on 2% agarose gels. Real time PCR reactions were
performed using the iQ SYBR Green Supermix (Bio-rad, Hercules, CA, USA). ROX
Passive reference Dye (Bio-rad, Hercules, CA, USA) 3.3 µl/ 1.25 ml of iQ SYBR
Green Supermix was added. Briefly, 3 µl cDNA, 0.8 µl of each primer (10 µM), 5.4 µl
RNase free water, 10 µl iQ SYBR Green Supermix were mixed to 20 µl final reaction
volume. Reaction conditions were: denaturation at 95oC for 10 min followed by 40
cycles of denaturation at 95oC for 15 s and annealing/elongation at 60oC for 60 s
followed by dissociation stage (95oC for 15 s, 60oC for 15 s followed by a slow ramp
to 95oC). All reactions were run in duplicate. The expression of gene of interest was
calculated in relation to HPRT. Relative expression ratios were calculated as R =
2[Ct(HPRT/-actin)_Ct(test gene)].
51
3.2.7 Histology
Placentas were fixed by immersion in 4% paraformaldehyde (Applichem, Darmstadt,
Germany). After tissue processing and embedding in paraffin, 5 µm sections were
prepared. Paraffin sections were stained with hematoxylin-eosin (H&E) and scanned
with the Mirax Midi Slide Scanner microscope (Zeiss, Jena, Germany).
3.2.8 Immunoblotting
Placentas, stored at -80°C, were homogenised in ice cold homogenisation buffer
(0.27 M sucrose, 2 mM EDTA, 0.5% NP40 prepared in 1x buffer A supplemented
with protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany)
(buffer A: 0.6 M KCl, 150 mM NaCl, 150 mM HEPES, pH 7.5,). Homogenates were
overlaid on a sucrose cushion (30% w/v sucrose, 2 mM EDTA, pH 8 prepared in 1x
buffer A) and centrifuged at 3,000 rpm for 10 mins at 4°C. After centrifugation,
supernatant obtained were stored as a cytoplasmic fraction, and pellets of nuclei
were resuspended in 1x nuclei extraction buffer (20 mM HEPES, pH 7.5, 400 mM
NaCl, 1 mM EDTA, pH 8) and incubated for 15 minutes on ice with intermittent
vortexing. Solubilised nucleated pellet fractions were centrifuged again at 15,000
rpm for 5 min at 4°C and supernatant collected as nuclear protein fraction. Protein
concentration was measured with a protein assay kit (Bio-rad protein assay, Munich,
Germany). 60 to 80 µg of proteins were solubilised in 5X Laemmli sample buffer and
run on 8% polyacrylamide gels. Briefly, protein was separated by SDS-PAGE,
transferred on nitrocellulose membrane by the wet transfer method and incubated
overnight with the following primary antibodies: rabbit polyclonal anti-HIF1 (1:1000,
Novus Biologicals) and mouse monoclonal -actin (1:10,000, Sigma). After washing,
nitrocellulose membranes were incubated with anti-rabbit secondary fluorescent
antibody (goat anti-rabbit IRdye 800 1:10,000, or goat anti mouse IRDye 680,
1:20,000, Li-COR, Nebraska, USA). After washing the membranes three times, the
protein signal was detected by scanning the membrane with the Odyssey Infrared
Imaging System (Li-COR Biosciences). All images were analyzed using analysis
software provided with the system to calculate the protein of interest/actin ratio.
52
3.2.9 Statistical analysis
Results are expressed as mean ± SEM. All data were tested for significance using
paired or unpaired Student’s test or one way ANOVA analysis followed by Tukey’s
multiple comparison and Bonferroni’s post test where appropriate. P<0.05 was
considered statistically significant.
53
4 RESULTS
4.1 Aldosterone dependent and independent regulation of renal K +excretion
4.1.1 Response to high K+ loading
To assess the tolerance of AS-/- mice to high K+ load, mice were given different K+
diets. On control (0.8% K+) and on 2% K+ and 3% K+ diets, AS-/- mice ingested
slightly, but not significantly more food than AS+/+ mice. With 5% K+ diet food intake
significantly decreased in AS-/- compared to AS+/+ mice (figure 18A). AS-/- mice drank
significantly more water compared to AS+/+ on control (5.1 ± 0.3 ml vs. 4.3 ± 0.2 ml, p
= 0.04), 2% K+ diet (8.0 ± 0.6 ml vs. 6.4 ± 0.4 ml, p = 0.04), and on 3% K+ diet (8.4 ±
0.7 ml vs. 5.5 ± 0.4 ml, p = 0.002). On 5% K+ diet, water intake was significantly
decreased in AS-/- mice compared to AS+/+ mice (6.3 ± 1.0 ml vs. 9.9 ± 0.8 ml, p =
0.02). As expected, in AS-/- mice urinary volume per 24 h was elevated already on
control diet and was significantly higher on 2% K+ and 3% K+ diet compared to AS+/+
mice on the corresponding diets. On 5% K+ diet, urinary volume was significantly
decreased in the AS-/- mice reflecting the diminished water intake (figure 18B).
Plasma Na+ concentrations revealed no difference among the genotypes on different
K+ diets. In contrast, plasma K+ concentrations on 2% K+ and 3% K+ diets was
slightly elevated in knockout mice and culminating to a hyperkalemic situation in AS-/mice on 5% K+ diet with significantly higher plasma K+ levels than the corresponding
AS+/+ mice (figure 18C and 18D). To examine the ability of the kidney to adapt to
changes in dietary K+ load, 24 h urinary electrolyte concentrations was measured by
ion chromatography. Urinary Na+ excretion of AS-/- mice was slightly elevated on
control and significantly higher on 2% K+ diet, but there was no difference on 3% K+.
On 5% K+ diet, AS-/- showed a lower Na+ excretion than AS+/+ mice. Urinary K+
excretion parallels the observations seen for urinary Na + excretion. AS-/- mice were
able to excrete K+ in the urine on control diet, and even excreted significantly higher
K+ on the 2% K+ diet compared to AS+/+ mice. However, on 3% K+ diet urinary K+
excretion in AS-/- mice was similar as in AS+/+ mice, and on 5% K+ diet, K+ excretion
was significantly decreased in AS-/- mice indicating their inability to adapt to very high
K+ load (figure 18E and 18F).
54
A
B
AS+/+
AS-/-
**
7
Urine Volume (mL/24h)
Food Intake (g/24h)
6
5
4
3
2
1
0
Control
2% K
3% K
+
5% K
3% K+
5% K+
3
2
1
175
7
150
6
Plasma K+ (mM)
Plasma Na+ (mM)
2% K+
4
Control
*
D
125
100
75
50
25
0
5
4
3
2
1
Control
2% K+
3% K+
0
5% K+
E
Control
2% K+
3% K+
5% K+
F
**
**
300
200
100
0
Control
2% K+
3% K+
5% K+
Urinary K+ excretion (mol/24h)
Urinary Na+ excretion (mol/24h)
**
5
+
C
**
6
0
+
**
***
4000
*
3000
2000
1000
0
Control
2% K+
3% K+
5% K+
Figure 18. Response of AS+/+, wild type mice, (open bars); and AS-/-, aldosterone
synthase-deficient mice, (filled bars) to control (0.8% K+), 2% K+, 3% K+, and 5% K+
loading. Mice were kept in metabolic cages and urine and blood electrolytes were
measured. Physiological responses in relation to food intake over 24 h (A), urine excretion
over 24 h (B), blood Na+ concentration (C) and blood K+ concentration (D) at the end of
experiment after 2 days of diet, urine Na+ excretion over 24 h (E) and urine K+ excretion
over for 24 h (F) are shown. Measurements were performed after 2 days of K+ diet. n = 5-7
mice per group. Values are mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
55
4.1.2 Effect of high K+ diet on distal nephron K+ and Na+ channels: Increased
apical expression of ROMK and maintained ENaC cleavage in AS -/- mice on 2%
K+ diet
In mice on 0.8% K+ diet, immunoblots showed significantly decreased protein
abundances for the uncleaved (90kDa) band of
significant difference in protein abundance for
-ENaC in AS-/- mice, and no
- and -ENaC between genotypes
(figure 19).
0.10
-/-
ENaC
90kDa
ENaC
35kDa
actin
*
0.08
43kDa
 ENaC/actin
+/+
0.06
0.04
0.02
0.00
+/+
-/-
+/+
-/-
90kDa
35kDa
+/+
-/-
0.6
-/-
ENaC
85kDa
actin
43kDa
 ENaC/actin
+/+
0.4
0.2
0.0
ENaC
actin
-/85kDa
70kDa
43kDa
 ENaC/actin
+/+
0.8
0.6
0.4
0.2
0.0
+/+
-/-
85kDa
+/+
-/-
70kDa
Figure 19. Protein abundance of ENaC in AS+/+ and AS-/- mice on 0.8% K+ (control) diet.
Immunoblots for -, - and -ENaC subunit in total membrane fractions from mouse kidneys.
All membranes were reprobed for -actin and all data were normalized against -actin. Bar
graphs summarizing data from immunoblotting. n = 5 mice per group. +/+, wild type mice;
-/-, aldosterone synthase-deficient mice. Values are mean ± SEM. * p ≤ 0.05.
56
The physiological data suggested that the AS-/- mice are able to adapt to a
moderate increases in dietary K+ intake, but cannot stand a high dietary K+ load (5%
K+). Therefore, we concentrated in the following experiments mainly on the 2% K+
diet and elaborated in more detail the aldosterone-dependent and independentmechanisms of the homeostatic adaptation of a dietary K+ load.
First, we examined the regulation and expression of the ROMK in response to
a dietary K+ load. Immunoblotting showed no significant difference in protein
abundance for the glycosylated and the non-glycosylated forms of ROMK on 2% K+
diet. Immunostainings of kidney sections from 2% K+ diet mice showed ROMK
localized to the apical membrane in PCs of the DCT and CNT in AS+/+ mice.
Interestingly, in AS-/- mice, there was also apical localization of ROMK in the DCT
and CNT with apparently stronger ROMK staining intensity compared to AS+/+ mice
(figure 20). We also checked for an altered expression of the BK channel at protein
level by using an antibody against the
-subunit. The BK channel could be only
detected in brain samples, but was not detectable in kidney samples (figure 21A).
We also examined the mRNA expression of the BK channel 1, β and 4 subunits
in kidneys from mice of both genotypes (figure 21B). All subunits were weakly
expressed in the kidney and no significant differences in the mRNA expression of BK
subunits were revealed between AS+/+ and AS-/- mice.
To test for the protein abundance of ENaC after 2% K+ diet loading,
immunoblotting for the different ENaC subunits was done on total kidney proteins
from AS+/+ and AS-/- mice
(figure 22A). Protein abundance for
-ENaC, both
uncleaved (95 kDa) form and cleaved (30 kDa) form, was not significantly different
between genotypes indicating maintained
-ENaC cleavage in AS-/- mice.
Immunoblots for -ENaC showed significantly increased protein abundance in AS-/mice compared to AS+/+ mice. Protein abundance for the uncleaved (85 kDa) form of
-ENaC in AS-/- mice was significantly increased compared to AS+/+ mice but did not
reveal difference in protein abundance for the cleaved (70 kDa) form of -ENaC
between the genotypes, again indicating maintained cleavage. These results
suggest increased protein abundance for - and - (85 kDa, uncleaved bands) ENaC
and maintained - and -ENaC cleavages in AS-/- mice.
57
Immunohistochemistry was used to determine whether the 2% K+ diet altered
the segmental and/or subcellular distribution of -, - and -subunits of ENaC in AS-/and AS+/+ mice (figure 22B). In the CNT, -ENaC staining was localized to the apical
membrane in AS+/+ mice and, interestingly, also in AS-/- mice whereas in the CCD, ENaC was localized to the apical membrane in few cells in both genotypes. In AS +/+
mice,
- and -ENaC staining was localized to the apical membrane in the CNT,
whereas staining in the CCD was mostly cytoplasmic. Interestingly, in AS-/- mice, and -ENaC staining was mostly cytoplasmic in the CNT and CCD. Nevertheless, in
parallel with the western blot results, the signal intensity of - and -ENaC staining
was stronger in AS-/- mice. Thus, AS-/- mice show an increased abundance of - and
-ENaC protein, but these ENaC subunits are mostly cytoplasmically localized. On
5% K+ diet, ROMK was localized to the apical membrane both in AS +/+ and AS-/mice. In AS+/+ mice -ENaC was localized to the apical membrane but was mostly
cytoplasmic in AS-/- mice (figure 24).
58
+/+
-/55kDa
ROMK
42kDa
actin
43kDa
+/+
-/-
ROMK
DCT
CNT
Figure 20. Expression and subcellular distribution of the renal outer medullary
potassium channel (ROMK) during 2% K+ loading in mouse kidney. (A) Immunoblotting
for mature glycosylated (55 kDa) and core glycosylated (42 kDa) ROMK channel in total
membrane fractions of kidneys. The membrane was reprobed for
-actin. n = 5 mice per
group. (B) Immunolocalization of ROMK channels in distal convoluted tubule (DCT) and
connecting tubule (CNT) in kidney sections. +/+, wild type mice;
-/-, aldosterone synthase-
deficient mice.
59
A
B
2^(Ct HPRT- Ct BK 1)
0.08
0.06
0.04
0.02
0.00
+/+
-/-
+/+
-/-
Only
antimouse AP
Sec. antibody
Primary BK
channel antibody
+ Sec . antibody
2^(Ct HPRT- Ct BK 2)
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
2^(Ct HPRT- Ct BK 4)
0.005
0.004
0.003
0.002
0.001
0.000
+/+
+
-/+
Figure 21. Expression of Ca activated large conductance K channel (BK) channel
during 2% K+ loading. (A) Immunoblotting for BK channel protein in total membrane
fractions from mouse kidney and brain samples with or without primary antibody. (B) Real
time PCR was used to assess the mRNA expressions of
1,
β and
4 subunits of BK
channel. n = 6 mice per group.
60
-/-
0.04
ENaC
90kDa
ENaC
35kDa
actin
43kDa
0.03
0.02
0.01
0.00
+/+
-/-
-/85kDa
actin
43kDa
-/-
35kDa
*
0.4
ENaC
+/+
90kDa
 ENaC/actin
+/+
 ENaC/actin
+/+
A
0.2
0.0
+/+
-/-
*
0.20
85kDa
70kDa
ENaC
actin
43kDa
 ENaC/actin
+/+
-/-
0.15
0.10
0.05
0.00
+/+
-/-
85kDa
B
+/+
ENaC
-/-
+/+
ENaC
+/+
-/-
70kDa
ENaC
-/-
+/+
-/-
CNT
CCD
Figure 22. Expression and subcellular localization of the epithelial sodium channel
(ENaC) during 2% K+ loading in mouse kidney. (A) Immunoblotting for -, - and -ENaC
subunits in total membrane fractions. All membranes were reprobed for -actin and all data
were normalized against -actin. Bar graphs summarizing data from immunoblotting. n = 5
mice per group. (B) Immunolocalization of
-,
- and -ENaC subunits in the connecting
tubule (CNT) and cortical collecting duct (CCD) in kidney sections. +/+, wild type mice;
-/-,
aldosterone synthase-deficient mice. Values are mean ± SEM. * p ≤ 0.05.
61
On 5% K+ diet (figure 23), -ENaC protein abundance for the uncleaved (90
kDa) and cleaved (30 kDa) bands, was significantly lower in AS-/- than in AS+/+ mice.
No significant difference in protein abundance for the -ENaC subunit was observed,
whereas
-ENaC protein abundance for the uncleaved (85 kDa) band was
significantly increased and for the cleaved (70 kDa) band significantly decreased in
AS-/- mice. ROMK protein abundance (glycosylated and nonglycosylated) was not
different between the genotypes. These results indicate decreased ENaC cleavage
in AS-/- after 5% K+ loading.
-/-
ENaC
0.20
90kDa
ENaC
ENaC/actin
+/+
35kDa
***
0.15
0.10
***
0.05
0.00
+/+
-/-
+/+
-/-
90kDa
35kDa
+/+
-/-
0.6
-/-
ENaC
0.5
85kDa
 ENaC/actin
+/+
0.4
0.3
0.2
0.1
0.0
-/85kDa
70kDa
ENaC
 ENaC/actin
+/+
0.20
*
0.15
***
0.10
0.05
0.00
+/+
-/55kDa
ROMK
42kDa
actin
+/+
-/-
70kDa
1.5
43kDa
ROMK/actin
+/+
-/-
85kDa
1.0
0.5
0.0
+/+
-/-
55kDa
+/+
-/-
42kDa
Figure 23. Expression of ENaC and ROMK in mouse kidneys during 5% K+ loading. (A)
Immunoblots for -, - and -ENaC subunits and ROMK in total membrane fractions from
mouse kidneys. All membranes were reprobed for
-actin and all data were normalized
against -actin. Bar graphs summarizing data from immunoblotting. n = 5 mice per group.
Values are mean ± SEM. * p ≤ 0.05, *** p ≤ 0.001.
62
+/+
-/-
ENaC
ROMK
Figure 24. Subcellular distribution of ENaC and ROMK in mouse kidneys during 5% K+
loading. Immunolocalization of
-ENaC and ROMK in mouse kidney sections.
-ENaC
staining is mostly localized to apical membranes in wild type (+/+) mice whereas in
aldosterone synthase-deficient mice (-/-) staining is mostly cytoplasmic. ROMK staining is
localized to apical membranes in both +/+ and -/- mice.
63
4.1.3 Functional ENaC and apical localization of ENaC in the late DCT in AS -/mice on 2% K+ diet
To determine in vivo the functional activity of ENaC, mice were treated with
amiloride, a specific blocker of ENaC. Urinary Na+ excretion was significantly higher
in the amiloride-treated group compared to vehicle-treated group in AS+/+ mice as
well as in AS-/- mice indicating active ENaC channels in both AS+/+ and AS-/- mice
(figure 25C). Furthermore, immunohistochemistry showed an apical localization of and -ENaC in the late DCT to but a mostly cytoplasmic localization of the channel in
the CNT and CCD in AS-/- mice (figure 25A). These results indicate that in AS-/- mice,
there may be functionally active ENaC channels in the kidney and they are mainly
located in the late DCT. To further confirm the functional activity of ENaC in the late
DCT, ex vivo patch clamping experiments were performed for ENaC (figure 25B) on
isolated, split-open DCT2, CNT and CCD segments from AS+/+ and AS-/- mice kept
on 2% K+ diet. Consistent with the results from immunohistochemistry, similar wholecell-amiloride-sensitive-currents were observed in the early ASDN segments of AS+/+
and AS-/- mice.
4.1.4 Impaired activation of colonic Na+ and K+ channels in AS-/- during 2% K+
loading
We also examined if similar aldosterone-independent functional ENaC channels
were present in distal colon. Barium-sensitive K+ channel currents and amiloridesensitive Na+ channel currents (figure 26) were significantly increased in AS+/+ mice
after 4 days of 2 % K+-loading, but absent in AS-/- mice. This indicates that
aldosterone, unlike in the kidney, is essential for the regulation for ENaC in response
to K+-loading in the distal colon.
64
A
B
ENaC
+/+
ENaC
CNT
-/-
CNT
DCT
DCT
mmol Na/mmol creatinine
C
0.9% NaCl
Amiloride
150
100
***
***
50
0
+/+
-/-
Figure 25. Functional activity and subcellular distribution of ENaC in kidney colon
during 2% K+ loading. (A) Immunolocalization of the ENaC - and -subunits in the late
distal convoluted tubule (DCT) and connecting tubule (CNT) in kidney sections from
aldosterone synthase-deficient (AS-/-) mice. (B) Amiloride sensitive whole cell Na+ channel
currents in the early aldosterone sensitive distal nephron (ASDN) from AS+/+ and AS-/- mice
on 2% K+ diet, n = 7-9 mice per group. (C) Urinary Na+ excretion after 4 h of vehicle or
amiloride injection. N = 8-11. Values are mean ± SEM. *** p ≤ 0.001.
*
-5
-10
-15
+/+
-/-
80
Amil  Isc (A cm-2)
Ba2+  Isc (A cm-2)
0
40
20
0
-20
0
2
Days
4
*
60
+/+
-/-
0
2
4
Days
Figure 26. Functional activity of ENaC and BK channel in distal colon during 2% K+
loading. Ussing chamber recordings of barium sensitive K+ channel and amiloride sensitive
Na+ channel currents in the distal colon. N = 5-6 mice per group. Values are mean ± SEM. *
p ≤ 0.05.
65
4.1.5 Role of angiotensin II in AS-/- mice
Mice were treated with losartan, an antagonist for the angiotensin type 1 receptor
(AT1R) to get insights in the role of angiotensin II for the rather compensated
phenotype of AS-/- mice, which is evident even on a high K+ (2% K+) diet. Food intake
was significantly decreased in losartan treated AS-/- mice compared to vehicle
treated AS-/- animals (3.1 ± 0.4 g vs. 5.5 ± 0.2 g, P = 0.0015) indicating food
avoidance after losartan treatment, whereas no difference in food intake was
observed in losartan vs. vehicle treated AS+/+ mice (5.5 ± 0.6 g vs. 5.3 ± 0.2 g, P =
0.8). No significant difference was seen in water intake in losartan vs. vehicle treated
AS-/- mice (2.6 ± 0.3 ml vs. 2.2 ± 0.2 ml, P= 0.4) as well as in AS+/+ mice (1.4 ± 0.1 ml
vs. 1.0 ± 0.1 ml, P = 0.06). Urinary Na+ excretion was significantly decreased in
losartan-treated AS-/- mice compared to vehicle-treated animals. K+ excretion in urine
was also significantly decreased in the losartan-treated AS-/- mice. In contrast, no
significant difference in urinary Na+ and K+ excretion was observed in losartan
treated vs. vehicle treated AS+/+ mice (figure 27A). Hyponatremia was seen in the
losartan treated AS-/- mice as shown by significantly decreased plasma Na +
concentration compared to vehicle treated mice. Interestingly, losartan treated AS -/mice were hyperkalemic as shown by significantly higher plasma K + concentrations
compared to vehicle treatment (figure 27B). Immunohistochemistry showed apical
localization of ENaC in vehicle-treated mice but mostly cytoplasmic after losartan
treatment in kidney sections from AS-/- mice (figure 27C).
66
A
Urinary Na excretion
Urinary K excretion
150
*
10
K/Cre (mmol/mg/dl)
Na/Cre(mmol/mg/dl)
15
*
5
0
Veh.
Losa.
Veh.
+/+
B
100
50
0
Losa.
Veh.
-/-
Losa.
Veh.
+/+
Plasma Na
Losa.
-/Plasma K
160
15
*
150
**
***
*
K (mmol)
155
Na (mmol)
*
145
140
10
*
5
135
130
Veh.
Losa.
+/+
C
Veh.
0
Losa.
-/-
Veh.
Losa.
+/+
Veh.
Losa.
-/-
ENaC
Veh. (-/-)
Losa. (-/-)
Figure 27. Effect of angiotensin type 1 receptor (AT1R) inhibition by losartan during
2% K+ loading on Na+ and K+ concentrations in blood and urine. (A) Urinary Na+ and K+
excretion in mice after 13 h of vehicle or losartan treatment. (B) Blood Na+ and K+
concentrations in mice after 13 h of vehicle or losartan treatment. n = 5, losartan group;
n = 4, vehicle group. (C) Immunolocalization of the -ENaC subunit in the early ASDN from
vehicle and losartan treated mice. +/+, wild type mice;
-/-, aldosterone synthase-deficient
mice. Values are mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
67
4.1.6
Decreased sodium chloride co-transporter (NCC) protein expression
and activity in AS-/- mice on 2% K+ diet
Next, we tested the expression and functional activity of NCC as the Na+
reabsorption by NCC along the DCT may affect or modulate the Na+ and K+ transport
systems in further downstream nephron portions. The results from qPCR showed no
significant difference in NCC mRNA expression between the genotypes (figure 28A).
In contrast, immunoblots, for total NCC and for phospho-specific forms of NCC,
namely
phosphothreonine
53
(pT53),
phosphothreonine
58
(pT58)
and
phosphoserine 89 (pS89), showed significant decreases in NCC protein abundance
and phosphorylation in AS-/- mice compared to AS+/+ (figure 28B).
To assess the in-vivo functional relevance of the reduced NCC abundance
and phosphorylation, mice were injected either with DMSO as vehicle or with the
diuretic hydrochlorothiazide to inhibit NCC. Urine was collected and analyzed for ion
excretion. Urinary Na+ excretion was significantly higher in hydrochlorothiazide
treated vs. DMSO treated AS+/+ mice as well as in AS-/- mice, but in line with the
immunoblot results, urinary Na+ excretion in hydrochlorothiazide-treated AS-/- mice
was significantly lower than in hydrochlorothiazide-treated AS+/+ mice (figure 28C).
4.1.7 Similar deoxycoticosterone levels in AS-/- mice on control and 2% K+ diet
To check for the possibility that increased deoxycorticosterone levels in AS-/- mice
might activate the MR, 11-deoxycorticosterone levels were measured in urine
samples (figure 29). No significant difference in the concentration of 11deoxycorticosterone levels was observed on control diet between AS+/+ and AS-/mice. During 2% K+ diet 11-deoxycorticosterone levels were significantly decreased
in AS+/+ mice compared to AS-/- mice. The 11-deoxycorticosterone levels were not
significantly different in AS-/- mice on control and 2% K+ diet.
4.1.8 AS-/- mice can concentrate urine similar to AS+/+ mice
AS-/- mice excrete more urine than AS+/+ mice. Therefore we checked for protein
abundance and subcellular localization of AQP2 in kidneys. In AS+/+ mice, AQP2 is
localized to the apical membrane whereas in AS-/- AQP2 is mostly cytoplasmic
(figure 30A). Protein abundance for AQP2 was increased in AS-/- mice as seen in
68
immunoblots (figure 30B). Urine osmolarity was significantly increased in AS+/+ as
well as in AS-/- after DDAVP treatment (figure 30C). The plasma Na+ levels were
significantly decreased in AS+/+ and AS-/- mice after DDAVP treatment. In contrast,
no significant change in levels of plasma K+ was seen in AS+/+ and AS-/- mice after
DDAVP treatment.
69
8
6
4
2
0
+/+
Urinar Na+ Excretion
C
NCC
Na / cre (mmol / mg / dl)
NCC (2^(HPRT-NCC)
A
-/-
60
***
***
40
***
20
0
(DMSO)
(HCTZ)
+/+
+/+
0.5
-/140kDa
NCC
actin
43kDa
NCC/actin
B
-/140kDa
actin
43kDa
0.3
0.2
0.1
-/-
43kDa
pT58 NCC/actin
actin
0.6
0.4
0.2
actin
-/140kDa
43kDa
-/*
0.6
0.4
0.2
+/+
-/*
0.10
pS89 NCC/actin
pS89 NCC
+/+
0.8
0.0
+/+
-/-
*
1.0
140kDa
pT58 NCC
+/+
0.8
0.0
+/+
*
1.0
pT53 NCC/actin
pT53 NCC
(HCTZ)
-/-
0.4
0.0
+/+
(DMSO)
0.08
0.06
0.04
0.02
0.00
+/+
-/-
Figure 28. Expression and in vivo functional activity of the sodium chloride cotransporter (NCC) in mouse kidneys during 2% K+ loading. (A) Real-time RT-PCR was
used to assess NCC mRNA levels in mouse kidneys. n = 7 mice per group. (B)
Immunoblotting for total NCC and phosphorylated forms of NCC at residues threonine 53
(pT53 NCC), threonine 58 (pT58 NCC) and serine 89 (pS89 NCC) normalized against
-
actin in total membrane fractions from mouse kidneys. Bar graphs summarizing data from
immunoblotting. n = 5 mice per group. (C) Urinary Na+ excretion after 4 h of
dimethylsulfoxide (DMSO, vehicle) or hydrochlorthiazide (HCTZ) treatment. n = 8, DMSO
+/+ group; n = 9 for +/+ HCTZ, -/- DMSO and HCTZ group. Values are mean ± SEM. * p ≤
0.05, *** p ≤ 0.001.
70
11-deoxycorticosterone
1000
*
ng/24h
800
+/+
-/-
600
400
200
0
cont. diet
2 % K+ diet
Figure 29. Concentration of 11- deoxycoticosterone in urine samples from AS+/+ and
AS-/- mice. Control diet n = 5 for each group; 2% K+ diet n = 4 for AS+/+ mice and n = 7 for
AS-/- mice. Values are mean ± SEM. * p ≤ 0.05.
71
A
+/+
B
-/-
-/-
+/+
AQP2
35-40 kDa
25 kDa
actin
43 kDa
CCD
C
Urine osmolarity
Uosm (mOsm)
2500
IMCD
***
***
2000
1500
1000
500
0
Before
DDAVP
Before
+/+
DDAVP
-/-
D
Plasma Na
Plasma K
160
5
**
150
*
4
*
K (mmol)
Na (mmol)
155
*
145
140
3
2
1
Veh.
DDAVP
+/+
Veh.
DDAVP
-/-
0
Veh.
DDAVP
+/+
Veh.
DDAVP
-/-
Figure 30. Expression and subcellular distribution of aquaporin 2 (AQP2) water
channel and its response to desmopressin (1-deamino-8-D-arginine vasopressin
(DDAVP)) treatment on 2% K+ loading. (A) Immunolocalization of AQP2 water channel in
cortical collecting duct (CCD) and inner medullary collecting duct (IMCD) in kidney sections
from mice. (B) Immunoblot of AQP2 protein and -actin as a loading control showing protein
abundance in total membrane fractions from whole kidneys. (C) Urine was collected before
and 4 h after DDAVP treatment from mice on 2% K diet and osmolarity was measured.
Urinary osmolarity is increased in both +/+ and -/- mice after DDAVP treatment. n = 5 mice
per group (D) Blood Na+ and K+ concentration after 4 h of vehicle or DDAVP treatment.
Lower blood Na+ concentration in DDAVP treated mice compared to vehicle group and
unchanged K+ concentration. n = 5 mice per group. Values are mean ± SEM. * p ≤ 0.05, ** p
≤ 0.01, *** p ≤ 0.001.
72
4.2. Role of aldosterone in pregnancy
4.2.1 Absence of a maternal preeclamptic phenotype in AS-/- mice during
pregnancy
To test the hypothesis that aldosterone deficiency during pregnancy may contribute
to the pathogenesis of pre-eclampsia, we measured SBP and urinary protein
excretion in AS+/+ and AS-/- dams before and during pregnancy. In order to eliminate
the genotype of the fetuses as confounding factor, AS+/+ females were mated to AS-/males and AS-/- females to AS+/+ males producing only AS+/- offspring. Before and
during pregnancy, SBP was significantly higher in AS+/+ than in AS-/- dams (figure
31A). While blood pressure of AS+/+ remained constant at about 110 mmHg
throughout pregnancy, SBP in AS-/- dams decreased from about 105 mmHg at day 1
to about 90 mmHg at day 21. Importantly, there was no difference in urinary protein
excretion and no increase in AS+/+ and AS-/- mice during pregnancy (figure 31B).
Thus, AS-/- dams did not become hypertensive and did not develop proteinuria during
pregnancy.
4.2.2 Presence of a feto-placental phenotype in AS-/- mice on control diet
Mean litter size at birth was lowered in AS-/- dams compared to AS+/+ mice (3.0 ±
0.55 vs. 6.22 ± 0.66, respectively; p<0.005) representing a strong fetal phenotype
(figure 31C). To examine whether the reduced litter size at birth was due to
intrauterine death of fetuses, dams were sacrificed on gestational day 18 (E18) and
the number and weight of fetuses and placentas were measured. The mean litter
size was again smaller in AS-/- compared to AS+/+ dams (2.67 ± 0.54 vs. 7.13 ± 0.48,
respectively; p<0.0001) (Figure 26A). The reduction of the number of pups paralleled
the presence of dark and necrotic placentas in AS-/- mice (figures 32A and 32B).
Necrotic placentas were mostly observed in series next to each other but sometimes
also observed interspersed between healthy placentas. Necrotic placentas were not
seen in any AS+/+ dams on control diet.
73
A
+/+
-/-
Systolic BP (mmHg)
140
120
100
80
*
***
**
**
** * **
**** *
**
***
*****
60
-1
1
3
5
7
9
11
13
15
17
19
21
23
Gestation (d)
B
+/+
-/-
C
8
Litter Size at Birth (n)
Protein/creatinine
(mg/ml/mg/dl)
0.3
0.2
0.1
6
2
0
0.0
0
5
10
15
Gestation (d)
20
**
4
+/+
-/-
Figure 31. Systolic blood pressure, proteinuria and litter size on control diet. (A)
Systolic blood pressure measured during the course of pregnancy from day 0 to day 21 in
AS+/+ (n = 6-9 animals/time point) and AS-/- (n = 3-9 animals/time point) dams. (B) Urine
protein concentration normalized to creatinine measured from spot urine during pregnancy in
AS+/+ (n = 3) and AS-/- dams (n = 6). (C.) Number of pups counted on the day of delivery in
AS+/+ (n = 9) and AS-/- (n = 9) dams. Values are mean ± SEM. *P < 0.05, **P < 0.005, ***P <
0.001 vs. AS+/+.
74
***
8
Litter Size at E18 (n)
Necrotic Placentas at E18 (n)
A
6
4
2
0
+/+
-/-
5
4
3
2
1
0
+/+
-/-
B
+/+
Normal
placenta
Necrotic
placenta
+/+
-/-
1000
***
900
**
800
700
600
500
1
2
3
5
6
7
8
9
Litter Size (n)
Mean Placenta Weight at E18 (mg)
C
Mean Litter Weight at E18 (mg)
-/-
100
+/+
-/-
90
80
**
70
60
50
1
2
3
5
6
7
8
9
Litter Size (n)
Figure 32. Litter size, litter and placenta weights, and healthy and necrotic
placentas in AS+/+ and AS-/- dams on day 18 of pregnancy. (A) Number of fetuses
and necrotic placentas were counted in the uterus from AS+/+ (n = total number of fetuses
and placentas from 8 females) and AS-/- dams (n = total number of fetuses and placentas
from 15 females). (B) Photos of fetuses and placentas in the uterus from AS+/+ and AS-/dams and comparison of healthy and necrotic placenta. (C) Mean litter and mean placenta
weight vs. litter size in AS+/+ (n = 8 litters) and AS-/- dams (n = 12 litters). Values are mean ±
SEM. **P < 0.005, ***P < 0.001 vs. AS+/+.
75
When the mean litter and placenta weights were plotted against litter sizes
(figure 32C), it became evident that the data for the AS-/- mice were not only shifted
to smaller litter sizes but also to reduced litter and placenta weights. This is most
evident when litters of the same sizes in AS-/- and in AS+/+ are compared (i.e. litter
sizes of 5 and 7 pups in figure 32C). In contrast, in very small litters (n = 1-3),
placental weight appeared to be normal. Histological examination of the placentas
further confirmed the reduced size of placentas in AS-/- dams, but did also reveal that
most of the placentas were otherwise healthy with a similar structural organization of
decidua basalis, junctional zone, and labyrinth as in AS+/+ dams (figure 33A and 33B,
left and middle section). Only dark placentas from AS-/- dams showed severe
coagulative necrosis with lymphocyte infiltrations in all three layers of the placenta
(figure 33A and B, right section).
4.2.3 High salt diet does not increase intrauterine survival, but improves fetal
growth in AS-/- mice and lowered systolic blood pressure irrespective of the
presence of aldosterone
Based on the assumption that aldosterone deficiency in AS-/- mice might compromise
placental perfusion, we tested if replacing renal sodium losses by feeding a high salt
diet (5% NaCl) before and during pregnancy improve the SBP, the weight of
placentas and of pups, and the litter sizes. AS+/+ and AS-/- females were fed a high
salt diet starting 12 days before until the end of pregnancy. After 4 days on a high
salt diet, the SBP rose in both groups of mice, but this increase occurred in parallel
and hence AS-/- female mice continued to have lower SBP than AS+/+ female mice
(figure 34A). During the first days (days 5-7) of pregnancy, the SBP dropped
significantly in both AS+/+ and AS-/- dams, but the decrease of SBP was much more
pronounced in AS+/+ mice than in AS-/- mice. Therefore, the BP difference between
genotypes became very small (days 5-7) and was no longer significant on days 15 to
17. This is in contrast to the measurements in dams on standard chow, where a
significant difference in SBP was maintained throughout the entire pregnancy (figure
31A). In AS-/-, blood pressure lower towards the end of gestation irrespective of the
salt intake (figure 34B). In AS+/+ mice, the SBP did not drop significantly in pregnancy
on a normal salt diet. However, if a high salt diet was provided, the blood pressure
dropped reaching the lowest levels at the end of pregnancy (figure 34C).
76
A
LZ
JZ DB
LZ
JZ DB
DB
LZ
#
*
+/+
Normal
-/Normal
*
-/Necrotic
B
LZ
JZ
DB
LZ
JZ
DB
LZ
DB
*
#
Figure 33. Histology of placentas. (A) Cross section and H&E stain of a placenta from an
AS+/+ dam and healthy and necrotic placentas from AS-/- dams. (B) Enlarged section of the
part of healthy placenta. (C) Enlarged section of the part of the necrotic placenta. Bar = 2000
µM. DB = decidua basalis, JZ = junctional zone and LZ = labyrinth zone. * indicates
lymphocyte infiltration and # indicates coagulative necrosis.
77
Of interest, both a low and a high volume state led to a blood pressure
reduction in pregnancy with the latter being even more consistently throughout
pregnancy.
The high Na+ diet did neither increase the mean litter size nor decrease the
number of necrotic placentas in AS-/- dams (figure 35A). However, the mean weights
of litters and placentas were no longer different between the genotypes when litters
of the same sizes were compared (figure 35B). This contrasts with the standard salt
intake, when both mean litter and placenta weights were smaller in AS-/- than in AS+/+
dams (figure 32C). Thus high salt intake does not increase the survival rate of
fetuses, but appears to improve the intrauterine growth of the surviving pups.
The beneficial effect of a high salt intake was seen for both genotypes.
Although the 5% NaCl intake did not increase the mean placental weight, it
significantly improved the weight of the fetuses in AS+/+ and AS-/- dams (figure 36A
and 36B). Consistent to these differential effects on fetal and placental growth,
placental efficiency (ratio of fetal weight over placental weight) was significantly
increased in both AS+/+ and AS-/- dams on high salt diet compared to control diet
(figure 36C). Interestingly, placental efficiency was not significantly different between
AS+/+ and AS-/- dams, neither on control nor on high salt diet (figure 36C).
4.2.4 Expression of the hypoxia inducible factor HIF1 in the placenta
Pre-eclampsia is associated with altered expression of key anti- and pro-angiogenic
factors [109]. We did not detect any differences in the mRNA expression of HIF1
(figure 37A), VEGF (figure 37B), and sFlt (figure 37C) between healthy appearing
placentas from AS+/+ and AS-/- dams kept on either a control or a high salt diet. Since
HIF1
is mostly regulated at the post-translational level by stabilisation and
degradation of the protein, we performed immunoblots. HIF1
protein abundance
was significantly increased in healthy placentas from AS-/- dams on control diet
(figure 39A). This difference disappeared on high salt diet (figure 33B). HIF1 protein
was not detected in necrotic placentas (figure 39A).
78
A
+/+
-/-
130
Systolic BP (mmHg)
5 % NaCl
120
110
**
**
100
*
90
80
-17 to-15
-10 to-8
5-7
15-17
Gestation (d)
B
C
-/- (cont.)
-/- (5 % NaCl)
120
###
110
100
90
80
*
*
70
130
Systolic BP (mmHg)
Systolic BP (mmHg)
130
+/+ (cont.)
+/+ (5 % NaCl)
120
110
***
100
***
90
###
80
70
-10 to -1
5-7
Gestation (d)
15-17
-10 to-1
5-7
15-17
Gestation (d)
Figure 34. Effect of high salt diet on systolic blood pressure (SBP), litter size, litter
and placenta weight and necrotic placentas at day 18 of pregnancy (A) SBP was
measured at different time points (days) before and during pregnancy in AS+/+ (n = 6-8
animals/time point) and AS-/- (n = 7-10 animals/ time point). (B) In AS-/- mice, no differences
in blood pressure was observed upon salt loading in pregnancy. SBP compared between
control and high salt (5% NaCl) diet at different time points just before pregnancy (-4 to -1
day for control and -10 to -8 for high salt) gestational day 5 to 7 and 15 to 17. (C) In
pregnant wild-type mice, salt loading led to a significant drop in blood pressure. SBP
compared between control and high salt (5% NaCl) diet at different time points just before
pregnancy (-4 to -1 day for control and -10 to -8 for high salt) gestational day 5 to 7 and 15 to
17. Values are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001 vs. AS+/+ (Figure 25A) or
vs. control diet (Figure 25B, C), ###P < 0.005 vs. high salt (5% NaCl) at -10 to -1 days.
79
Necrotic Placentas at E18 (n)
Litter Size at E18 (n)
8
6
4
2
0
+/+
-/-
6
*
4
2
0
+/+
-/-
1600
+/+
-/-
1400
1200
1000
800
600
1
2
3
4
5
6
Litter Size (n)
7
8
Mean Placenta Weight at E18 (mg)
Mean Litter Weight at E18 (mg)
B
150
+/+
-/-
100
50
0
1
2
3
4
5
6
7
8
Litter Size (n)
Figure 35. Effect of high salt diet on systolic blood pressure, litter size, litter and
placenta weight and necrotic placentas at day 18 of pregnancy. (A) Number of fetuses
and necrotic placentas detected in AS+/+ (n = 6) and AS-/- dams (n = 9) at day 18 of
pregnancy. (B) Mean litter and mean placenta weight vs. litter size in AS+/+ (n = 6) and AS-/dams (n = 9). Values are mean ± SEM. *P < 0.05 vs. AS+/+.
80
Mean Litter Weight at E18 (mg)
1500
1200
*** ** ***
900
600
300
0
2
5
6
7
8
9
Litter Size (n)
120
+/+ (cont.)
+/+ (5% NaCl)
100
80
***
***
60
40
2
5
6
7
8
9
Litter Size (n)
-/- (cont.)
-/- (5% NaCl)
**
1500
1200
Mean Placenta Weight at E18 (mg)
B
+/+ (cont.)
+/+ (5% NaCl)
Mean Placenta Weight at E18 (mg)
Mean Litter Weight at E18 (mg)
A
*
900
600
300
0
1
2
3
4
5
6
7
Litter Size (n)
120
-/- (cont.)
-/- (5% NaCl)
100
80
60
40
1
2
3
4
5
6
7
Litter Size (n)
Foetal weight /placenta weight
at E18 (mg/mg)
C
20
##
##
+/+
-/-
15
10
5
0
+/+
-/-
Control diet
5%NaCl
Figure 36. Comparison of litter and placenta weights, and placental efficiency between
control vs. high salt diet in AS+/+ and AS-/- dams. Mean litter weights (A) and mean
placenta weights (B) on control diet vs. high salt diet in AS+/+ (control diet n = 8 litters, high
salt diet n = 6 litters) and AS-/- dams (control diet n = 12 litters, high salt diet n = 9 litters). (C)
Placental efficiency in AS+/+ (control diet n = 8 litters, high salt diet n = 6 litters) and AS-/(control diet n = 13 litters, high salt diet n = 9 litters) dams on control and high salt diet.
Values are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.001 vs. control diet, ## P < 0.005
vs. AS+/+ on control diet, ***P < 0.001 vs. AS-/- on control diet.
81
A
2^(Ct HPRT- Ct HIF1)
4
control diet
5% NaCl
3
2
1
0
B
+/+
-/-
2^(Ct HPRT- Ct VEGF)
4
control diet
5% NaCl
3
2
1
0
C
+/+
-/-
2^(Ct HPRT- Ct sFlt-1)
4
control diet
5% NaCl
3
2
1
0
+/+
-/-
Figure 37. Expression of VEGF, sflt and HIF1 in placentas. mRNA expression of HIF1
(A) VEGF (B), and sFlt (C) in healthy placentas from AS+/+ (n = 5) and AS-/- (n = 4-5 ) dams
on control and high salt (5% NaCl) diet.
82
Control diet
A
***
1.5
1.5
2^ (Ct HPRT- Ct TNF )
2^(Ct HPRT- Ct MCP-1)
2.0
***
1.0
0.5
0.0
+/+
-/Healthy
1.0
***
0.5
0.0
-/-
+/+
Necrotic
-/-
-/-
Healthy
Necrotic
5 % NaCl diet
B
0.8
***
0.6
0.4
0.2
0.0
+/+
-/Healthy
**
1.5
2^(Ct HPRT- Ct TNF )
***
1.0
2^(Ct HPRT- Ct MCP-1)
***
-/-
*
1.0
0.5
0.0
+/+
Necrotic
-/Healthy
-/Necrotic
Figure 38. Expression of inflammatory markers MCP-1 and TNF in placentas. mRNA
expression of MCP-1 and TNF was tested by qPCR in healthy placentas from AS+/+ (n = 5),
and healthy and necrotic placentas from AS-/- (n = 5 each) dams on control (A) and high salt
(5 % NaCl) (B) diet. Values are mean ± SEM. *P < 0.05 and ***P < 0.001 vs. healthy
placentas from AS-/-, **P < 0.005 and ***P < 0.001 vs. AS+/+.
4.2.5 Expression of pro-inflammatory factors in necrotic placentas
MCP-1 and TNF
are key pro-inflammatory cytokines involved in inflammatory
processes. Expression of MCP-1 and TNF
mRNA was significantly increased in
necrotic placentas from AS-/- dams on control (figure 38A) and high salt diet (figure
38B) but not in healthy appearing placentas from both genotypes.
83
A
Control diet
HIF1
Actin
Hif1 /  actin
2.0
1.5
1.0
0.5
0.0
B
**
+/+
-/-
+/+
-/-
5 % NaCl diet
HIF1
Actin
Hif1 /  actin
2.0
1.5
1.0
0.5
0.0
Figure 39. Protein expression of HIF1
+/+
was examined in placentas from AS
in placentas. The protein abundance of HIF1
and AS-/- dams on control (A) and high salt (5% NaCl)
(B) diet. Hypoxic and normoxic control samples are collected from cell culture as positive
and negative controls, respectively. Bar graph summarize the results from two independent
blots of n= 6 normalized to -actin measured on the same membranes. Values are mean ±
SEM. **P < 0.005 vs. AS+/+.
84
5 DISCUSSION
5.1 Role of aldosterone in renal potassium excretion
5.1.1 Adaptation to high potassium diet is possible without aldosterone
The traditional view of the control of K+ homeostasis assumes that increases in
plasma K+ concentrations (e.g. in response to a K+ rich meal), directly stimulate
aldosterone secretion by the adrenal glands. Then, the resulting enhanced plasma
aldosterone levels stimulate renal K+ secretion until plasma K+ return to normal. This
classical feed-back model has been challenged by studies of Rabinowitz and others
[176, 177], who showed that a K+ rich diet may provokes a kaliuretic response even
before plasma K+ and plasma aldosterone levels increase [178]. Moreover, they
showed that aldosterone has very weak kaliuretic activity at normal physiological
levels, and profoundly affects renal K+ excretion only when elevated to pathological
levels [176, 179]. Notably, also the well-known circadian variations of urinary K+
excretion do not match with the diurnal changes in plasma aldosterone and K+ [176].
Based on these observations, Rabinowitz proposed receptors in the gut, portal vein
or liver responsible for the diet-induced kaliuretic reflex and suggested that the CNS
and/or kaliuretic factors stimulate renal K+ secretion independent from plasma K+
and aldosterone. This would constitute a feed-forward control mechanism for K+
homeostasis, which would prime the kidney to excrete K+ to avoid high postprandial
peaks of plasma K+, with its possible detrimental effects on heart and neuronal
excitability. In fact recent studies in rat further supported the idea of the existence of
a gut-dependent feed-forward system that senses K+ intake during a meal and
rapidly enhances the renal clearance of plasma K+ [180]. In our experiments, we did
not test for the existence of a gut-dependent feed-forward system that regulates
renal K+ excretion. However, our data strongly support the idea of an aldosteroneindependent control system. Consistent with this observation, we found increased
urinary K+ excretion and adaptation in AS-/- mice within a physiologically K+-rich diet
(2% K+ diet). The increased urinary K+ excretion might be a result of combined
effects of feed-forward and feedback mechanisms. Decompensation of the system
occurred at unphysiologically high K+-intake (5% K+ diet). The food aversion in AS-/mice on this unphysiological 5% K+ diet were consistent with previous observations
in adrenalectomized rats on high K+ diet [181]. In this previous study
85
adrenalectomized rats with basal or 10x times higher aldosterone infusion consumed
all their diet independent of the amount of K+ supplementation. In contrast,
adrenalectomized rats with little or no aldosterone supplementation and given
access to the moderately or highly K+ supplemented diet became anorexic. Despite
the reduced diet intake, the adrenalectomized rats became hyperkalemic and some
of them eventually died either because of the anorexia or the hyperkalemia [181].
Consistent with these results, we found that AS-/- mice on 5% K+ diet were
hyperkalemic and lost body weights. The hyperkalemia and food aversion in this
experiment was likely related to a decreased capacity to secrete K+ secondary due
to the lack of aldosterone.
5.1.2 Aldosterone independent regulation of ROMK and ENaC
Previous studies on adrenalectomized animals indicated that a high K+ diet
increases renal Na+ and K+ channel activities independent from aldosterone [166,
167]. However, adrenalectomies in rodents are often incomplete. Moreover, a
physiologically relevant local renal aldosterone producing system appears to exist,
which becomes activated by angiotensin II and a low salt intake in adrenalectomized
rats [169]. Local synthesis of mineralcorticoids was also observed in other tissue
including the heart [182]. In AS-/- mice, any aldosterone production in adrenal and
extra-adrenal tissues is prevented due to the systemic disruption of the aldosteronesynthase gene. Thus, our experiments now conclusively demonstrate that a high K +
intake increases Na+ and K+ channel activities independent from aldosterone, at
least as long dietary K+ intake is increased to the physiologically upper limit of 2%
K+.
Apparently,
the
aldosterone
precursors
corticosterone
[9]
and
deoxycorticosterone (this study) are only marginally or not elevated in AS-/- mice
suggesting that there is no up-regulation of other members of the corticosteroid axis
that could compensate for the lack of aldosterone.
In line with previous studies that suggested aldosterone-independent
activation of Na+ and K+ channels in response to a high K+ diet [166, 167], we show
now at the molecular level a maintained K+-diet induced regulation of ENaC and
ROMK in aldosterone-deficient mice. In both AS+/+ and AS-/- mice the 2% K+ diet
caused an apical translocation of ROMK and a proteolytic activation of ENaC
subunits. Likewise, AS-/- mice showed the same natriuretic response to amiloride as
86
the AS+/+ mice and showed similar ENaC sodium currents in the early ASDN
segments as wild type mice. Immunohistochemistry revealed that in mice of both
genotypes the K+-diet induced activation of ROMK and ENaC predominantly
occurred in the early ASDN. Given the fact that aldosterone-binding and expression
of the mineralocorticoid-receptor appear to be homogenous along the entire ASDN
[28], the axial gradient along the ASDN of the response of ROMK and ENaC to a
high K+ diet does already suggest that aldosterone-independent factors significantly
contribute to the regulation of these two channels along the ASDN.
The axial gradient of ROMK and ENaC regulation is consistent with the idea
that the early ASDN is the most critical for the maintenance of Na+ and K+ balance.
In fact, patch clamp studies in on isolated ASDN segments of rats kept on a high K+
diet or treated with aldosterone indicated that ENaC activity is several times larger in
the CNT than in the CCD [183]. DCTs and CNTs of rats, rabbits and mice have
several fold higher Na+/+K-ATPase activity than the CDs of the respective species
[184]. Moreover, several regulatory proteins linked to hypertension or K+ excretion
such as WNK4, WNK1 and tissue kallikrein are localised in the DCT and CNT rather
than in the CD. Tissue kallikrein, a serine protease, is a kaliuretic factor involved in
renal K+ excretion after high K+ loads [185]. Earlier studies in collecting duct specific
-ENaC knockout mice showed the importance of the early ASDN (late DCT/CNT)
for the preservation of Na+ and K+ balance [186]. This study indicated the importance
of early ASDN (late DCT/CNT) in achieving Na+ and K+ balance and demonstrated
that expression of ENaC in CD is not a prerequisite to maintain Na+ and K+ balance
[186].
5.1.3 Angiotensin II dependent potassium excretion
AS-/- mice have very high levels of angiotensin II. AT1 receptors have been
demonstrated by immunohistochemical studies in DCTs, CNTs and CDs [187]. We
hypothesized that in the absence of aldosterone, the high angiotensin II levels might
be important to ensure renal K+ excretion. In fact, Inhibition of AT1 receptors for
angiotensin II by losartan led to a decompensation of AS-/- mice on high 2% K+ diet.
The mice became severely hyperkalemic and showed a clear avoidance for the K +
enriched food. The accompanying hyponatremia was unexpected, but might be due
to a combination of reduced food intake, normal water intake, and decompensated
87
renal
Na+
excretion.
Our
immunohistochemical
data
suggest
that
the
decompensation of the losartan-treated mice was related to a de-activation of ENaC
which diminished the driving force for renal K+ secretion along the ASDN. The role of
angiotensin II for ENaC regulation is supported by several previous studies. Long
term systemic infusion of angiotensin II increases -ENaC expression in rat kidney
cortex [188]. Likewise, AT1 receptor knockout mice have a reduced
-ENaC
expression despite elevated plasma aldosterone levels [189]. Angiotensin II was
shown to directly stimulate amiloride-sensitive sodium channel activity in isolated late
distal tubules and initial collecting ducts. The effect could be blocked by the luminal
administration of the AT1 receptor blockers candesartan or losartan [190, 191].
Furthermore, in type 2 diabetes and obesity inappropriate Na+ retention was
associated with AT1 receptor dependent activation of ENaC channels [192]. In
contrast to ENaC, ROMK and SK channel activity was proposed to be inhibited by
angiotensin II. During dietary K+ restriction, inhibition of AT1 receptors by losartan
augments apical K+ channel activity likely by interfering with Src family protein
tyrosine kinase (PTK) and mitogen activated protein kinase (MAPK) dependent
inhibition of ROMK and BK activity, respectively. However, this effect was only
evident during dietary K+ restriction. No inhibitory effect of angiotensin II on ROMK or
BK channel was observed during normal K+ diet [193, 194]. Consistently, in our
experiments on mice on standard and high K+ diet, we did not find any evidence for
an inhibitory effect of angiotensin II on ROMK. In fact, the AS-/- mice, which are
known to have exceedingly high angiotensin II levels [9], showed a rather prominent
apical ROMK localization that appeared to be even more pronounced than the one
seen in wild type mice.
Interestingly, losartan caused hyperkalemia and K+-food avoidance only in
AS-/-, but not in AS+/+ mice suggesting that AT1 inhibition is per se not sufficient to
significantly impair renal K+ handling and hence K+ homeostasis. Only when both
aldosterone and angiotensin II actions are lacking mice appear to decompensate.
Consistently, meta-analysis of clinical studies on patients with hypertension, heart
failure
and chronic kidney disease (CKD) demonstrated that the incidence of
hyperkalemia is
below 2% when only one component of the renin-angiotensin-
aldosterone system (RAAS) is inhibited, but increases to about 5-10% (depending on
the underlying disease) when two components of the RAAS are inhibited. In patients
88
with hypertension, the combination of an aldosterone receptor antagonist with an
angiotensin II receptor blocker was shown to increase serum K+ level to significantly
higher values than those seen in patients with a AT1 receptor blocker mono-therapy
[195].
5.1.4 Polyuria-dependent potassium excretion?
As previously reported [170], AS-/- mice are polyuric. This polyuria is seen already on
standard diet and becomes even more prominent on a high K + diet. Because the
activity of renal potassium channels such as maxi K/BK channels is flow-dependent,
we speculated that the enhanced diuresis may contribute to the maintained renal K +
secretion in AS-/- mice. However, lowering diuresis by injection of the synthetic
vasopressin analogue DDAVP did not lower urinary K+ excretion and did not
increase plasma K+ compared to wild type mice. Although we cannot exclude that
under these condition a vasopressin-dependent activation of K+ channels [196]
compensated for the lowered flow-dependent K+-channel activation, the data indicate
that a high urinary flow rate is at least not critical for K +-homeostasis in AS-/- mice.
Moreover, we could not detect any difference in the mRNA expression of BK channel
subunits
1,
β and
4 between wild type and AS-/- mice suggesting that the
aldosterone-deficiency did not lead to a compensatory up-regulation, at least at the
mRNA level, of flow-dependent K+-channels in the kidneys of AS-/- mice.
The reason for the polyuria in AS-/- is unclear. Theoretically, it could be the
result of an increased water intake due to enhanced thirst or due to renal water loss
due to an impaired urinary concentration mechanism caused by an insufficient
secretion of vasopressin from the pituitary (central diabetes insipidus) or caused by
an insufficient action of vasopressin on the kidney (renal diabetes insipidus). The
AS-/- deficient mice have exceedingly high angiotensin II levels and angiotensin II is
known to stimulate thirst [197]. However, previous data from Olivier Smithies group
showed that AS-/- mice, which were restricted to the same water intake as AS+/+
mice, still have a significantly lower urine osmolarity than wild type mice. This
suggests a renal concentration defect rather than an angiotensin II-induced
polydipsia as the cause for the observed polyuria [170]. Because it is technically very
challenging, we did not measure vasopressin levels in our mice, but the previous
studies from Olivier Smithies reported similar vasopressin levels in wild type and in
89
AS-/- mice suggesting that more likely a renal defect is responsible for the observed
polyuria. In fact, there is ample evidence that aldosterone increases vasopressin
mediated water transport in the renal collecting system [198, 199] either by direct
effects on renal water channels such as AQP3 [200] or by increasing ENaC activity
which may osmotically drive renal water reabsorption [201]. Consistent with a renal
water transport defect, our immunofluorescent studies revealed a slightly reduced
apical localization of the water channel AQP2 in the medullary collecting ducts of
AS-/- mice, though the total abundance of AQP2 protein (our study) and mRNA [170]
was found to be increased in AS-/- mice. Despite of the apparent mild trafficking
defect for AQP2, AS-/- mice are still responsive to vasopressin. In previous studies
with mice on standard diet as well as in our study on mice on 2% K + diet, the V2
receptor-specific agonist DDAVP increased similar urine osmolarity in both AS+/+
and AS-/- mice.
5.1.5 Aldosterone-independent down regulation of NCC contributes to K+
secretion
Previous studies showed that the thiazide-sensitive Na-Cl co-transporter is an
aldosterone-induced protein [202]. Consistent with these results, we found a reduced
total NCC and phospho-NCC abundance. NCC mRNA expression and the fractional
volume of the DCT was similar for mice of both genotypes indicating that the NCC
down-regulation occurs at the posttranscriptional level and is independent from a
major epithelial remodelling of the distal nephron. Consistent with the reduced NCC
phosphorylation, which is thought to be indicative for a reduced NCC activity [203],
the AS-/- showed a reduced thiazide-sensitive natriuresis when compared to AS+/+
mice. On a high K+ diet, NCC expression and phosphorylation decreased in mice of
both genotypes indicating that NCC-downregulation might be part of the homeostatic
response of the kidney to dietary K+ loading and that this effect is aldosteroneindependent.
Although the underlying mechanism for the K+-induced downregulation of
NCC is unclear, the functional consequences are likely important for the
maintenance of K+ balance. The decreased NCC activity augments Na+ delivery to
the early ASDN where then the increased luminal Na+ concentration increases the
driving force for Na+ reabsorption via ENaC which eventually fosters K+ secretion via
90
ROMK. A strong relationship between high Na+ delivery to the distal tubule and
increased K+ excretion is well established [22, 204]. Also patients with Gitelman’s
syndrome due to loss-of-function mutations in NCC tend to have an increased renal
K+
excretion
leading
to
hypokalemia
[205].
Conversely,
patients
with
pseudohypoaldosteronism type II, having an enhanced NCC activity due to
mutations in WNK kinases, develop hyperkalemia [206]. In these genetic diseases
not only NCC activity, but also plasma aldosterone levels are changed and it is likely
that both factors contribute to the altered renal K + handling. In the AS-/- mice, plasma
aldosterone levels are constantly set to zero and it appears that K + homeostasis in
these mice is largely achieved by adapting the Na + delivery to the early ASDN in
which ENaC is activated via angiotensin II.
5.1.6 Potassium induced activation of Na+ and K+ channels in colon is
aldosterone dependent
The distal colon contributes to K+ excretion and thus to K+ homeostasis. The distal
colon is an aldosterone-sensitive segment and aldosterone is responsible for
electrogenic Na+ reabsorption through ENaC channels in the distal colon [207]. Also,
aldosterone is well known to induce K+ secretion through BK channel in the distal
colon. Previous studies revealed increased mRNA expressions of BK and ENaC
channels in mouse distal colon on high K+ diet [97, 105]. Consistently, we also
demonstrated K+ induced activation of ENaC and BK channels in AS+/+ mice but not
in AS-/- mice indicating the necessity of aldosterone in the distal colon for stimulating
Na+ absorption and K+ secretion. Loss of distal colon K+ secretion activity in AS-/mice was compensated at least partially by increased K+ secretion in the kidney.
However, unlike Na+ dependent K+ secretion in the kidney, it is not clear if K+
secretion in the distal colon is a Na+ dependent process. One recent study
demonstrated that BK channels are primarily expressed in crypts and -ENaC mainly
expressed in surface cells of the distal colon. They also showed at the functional
level that ENaC mediated Na+ reabsorption and BK channels mediated K+ secretion
are two functionally independent processes [105]. In contrast, two studies with
radioactive tracer flux measurements in rats with in vivo or in vitro elevated plasma
aldosterone showed a ~40% reduction of serosal to mucosal tracer fluxes after
luminal amiloride application indicating reduced K+ secretion [104, 208]. This
91
indicates that a fraction of colonic K+ secretion may also occur in surface epithelial
cells under elevated aldosterone levels and uses Na + influx to drive this process or
that Na+ absorption generates a lumen-negative potential that favours K+-secretion
by crypt cells. A study using conductance scanning of epithelial surfaces concluded
that aldosterone induced K+ secretion resides in surface cells and K+ secretion
induced by adrenaline is present in both crypt as well as in surface epithelial cells
[208]. It is clear that aldosterone plays an important role in K + secretion in the distal
colon, although the exact mechanisms including its Na+ dependency are still not
clear.
Summary and perspectives
The final conclusion of this study is summarized in figure 40. In AS+/+ mice, on
control diet, the major part of the distally delivered Na+ is reabsorbed via NCC in the
DCT1 and DCT2 and a small remaining portion is reabsorbed via ENaC to maintain
fluid balance as well as K+ secretion. On the 2% K+ diet, the expression and activity
of NCC is decreased to increase Na+ delivery to the more distal nephron segments
to enhance K+ secretion. Therefore AS+/+ mice are able to maintain both fluid volume
and K+ secretion. Aldosterone plays an important role in ENaC regulation in AS+/+
mice. In contrast, in AS-/- mice, on control diet, NCC activity is already decreased
compared to AS+/+ mice and Na+ is reabsorbed only to some extent by NCC
transporters in DCT1 and DCT2 but more Na+ is delivered to the distal segments to
be absorbed by ENaC channels in the early ASDN to maintain both Na+ reabsorption
and K+ secretion. On the 2% K+ diet, a further decrease in NCC expression and
activity occurs to further increase Na+ delivery to the early ASDN to stimulate K+
secretion in order to excrete the excess K+ load. Angiotensin II might be playing an
important role for the activation of ENaC channels in the early ASDN in AS-/- mice in
the absence of aldosterone. On the 5 % K+ diet, residual ENaC activity only in the
early ASDN in AS-/- mice is not sufficient to maintain the secretion of the very high
load of K+ and ultimately mice show decompensation.
In summary, we describe the aldosterone-independent activation of ENaC
and ROMK in the early ASDN. The permissive effect of angiotensin II was important
for the residual activity of ENaC to excrete K+. On high K+ diet, the downregulation of
NCC is an important factor to increase Na+ delivery to the ASDN. We speculate that
92
the organism of the mice prefers to downregulate Na+ retaining mechanism on the
expense of a lower blood pressure rather than to risk hyperkalemia with all its
potentially deleterious effects on excitable tissues.
NCC
ENaC
ROMK
0.8% K
Na+
Na+
2% K
K+
Na+ K+
Na+
AS+/+
Na+ K+
?
DCT1
DCT2
Na+
Na+ K+
DCT1
DCT2
CNT
DCT1
Na+
DCT2
Na+ K+
MR
Aldo.
CNT
Na+ K+
AS-/-
Angiotensin II
CNT
DCT1
DCT2
CNT
Angiotensin II
Figure 40. Model to explain aldosterone dependent and independent regulation of
renal K+-excretion in AS+/+ and AS-/- mice on control and high K diet.
93
5.2 Role of aldosterone in pregnancy
Plasma volume expansion in the presence of high aldosterone levels is considered
crucial for healthy pregnancies, both of which are low in the most severe form of
hypertensive pregnancies, pre-eclampsia. In order to examine the role of
aldosterone in the development of pre-eclampsia, we used an aldosterone synthase
knockout mouse model (AS-/-). AS-/- mice have a compensatory increase of renin and
angiotensin II to supraphysiological concentrations but no aldosterone [9] allowing to
independently test the role of aldosterone in pregnancy. Additional effects of these
high renin and angiotensin II may occur but could not be tested because survival of
AS-/- mice depends on high angiotensin II levels and pharmacological blockade of
AT1 receptors is lethal in AS-/- mice (unpublished observations).
New-onset hypertension and proteinuria with feto-placental developmental
disturbances are considered to be cardinal features of pre-eclampsia [109]. We
found that AS-/- dams did not develop preeclampsia as suggested by the absence of
hypertension and proteinuria during pregnancy. A previous study measured SBP by
telemetry during pregnancy and showed no change in SBP during the first 5 days,
followed by a drop in SBP from days 6 to12, and again from day 15 to 17 of
pregnancy [209]. In our hands, using the less sensitive tail cuff method, we found
reduced SBP in wild type mice during pregnancy in response to salt loading. Though
contra-intuitive, this observation is in line with some clinical observations during salt
loading in pregnancy suggesting a beneficial systemic feedback on maternal
hemodynamics upon optimal placental perfusion [163, 164]. Thus, pre-eclampsia is
not caused by a lack of aldosterone alone, at least in mice.
However, the mouse model revealed a number of other interesting
observations demonstrating an important role of aldosterone in normal pregnancy.
AS-/- dams were hypotensive before pregnancy and became even more hypotensive
during mid to term pregnancy. The resulting feto-placental phenotype of a decreased
litter size and smaller or necrotic placentas without fetuses at term suggests a
severe placental hypoperfusion. The sum of intact fetuses and necrotic placentas in
AS-/- mice was similar to the average number of fetuses in AS+/+ dams, indicating
that AS-/- dams were able to conceive normally but had impaired placental
development and function, predisposing to intrauterine death. The few normal weight
94
placentas and fetuses observed in AS-/- dams carrying small litters would such
provide a residual blood and nutrient supply to the remaining placentas.
Histological examination of healthy placentas from AS-/- dams demonstrated
normal structural patterns but reduced size. One important reason for lower placental
weight might be the absence of direct effects of aldosterone on the placenta in AS-/dams. In vitro experiments have
revealed a direct role for aldosterone in the
modulation of trophoblast growth and placental size [5] and in vivo studies in ewes
suggested a role of aldosterone in placental growth [162]. Placental size in rats and
humans is positively correlated with plasma levels of aldosterone [5]. Thus,
aldosterone may positively modulate placental growth, similar to its proliferative
capabilities in organs such as the heart and the kidney [210, 211].
Reduced fetal weight in AS-/- dams might thus be due to placental
insufficiency. In normal human pregnancy, plasma volume expansion is associated
with increased plasma aldosterone level whereas both were decreased in
pregnancies
with
intrauterine
growth
restriction
and
pre-eclampsia
[146].
Pre-eclampsia is characterised by extracellular volume expansion but low
intravascular volume which is caused at least in part by a shift of fluid from the
intravascular to the interstitial fluid space due to vasoconstriction and leaky
endothelium [212-214]. Lack of aldosterone in AS-/- dams might thus critically impair
plasma volume expansion and thereby cause reduced uterine blood supply similar to
functional
aldosterone
deficiency
in
mice
chronically
exposed
to
the
mineralocorticoid receptor antagonist spironolactone during pregnancy which led to
reduced fetal umbilical blood flow [5]. Moreover, modelling a reduced uterine blood
supply by constriction of the uterine artery in rats from gestational day 14 caused
placental insufficiency resulting in low-birth weight [215].
One common cause for the decreased litter size, reduced fetal and placental
weight, and necrotic and inflammatory placentas in AS-/- dams could be hypoxia. In
rats, hypoxia during the last 11 days of pregnancy results in smaller sized litters,
decreased placental and fetal weights, and placental resorption [216]. Low oxygen
tension affects cytotrophoblast proliferation and invasion and ultimately fetal growth
[116]. Consistently, the hypoxia-induced factor HIF1, a master regulator in the
adaption to hypoxia, is essential for mammalian placentation [217]. HIF1
protein
95
abundance was significantly higher in AS-/- mice on control diet, but was reduced to
control levels on 5% NaCl diet in the normal appearing placentas suggesting hypoxia
and rescue by high NaCl intake. Consequently, AS-/- necrotic placentas showed
coagulative necrosis and severe lymphocyte infiltration and elevated levels of proinflammatory cytokines MCP-1 and TNF .
Aldosterone plays a central role in the long-term control of extracellular
volume by adapting renal and extrarenal reabsorption of sodium to blood pressure
[17]. Thereby, extracellular NaCl and volume is enlarged, blood pressure and organ
perfusion increased. AS-/- mice are hypotensive despite highly elevated levels of
renin and angiotensin II. High salt diet reverses renin and angiotensin II
abnormalities without increasing blood pressure indicating that the extracellular
volume status is at least partially normalized [218]. To rescue the possibly reduced
plasma volume and lower uterine blood flow in AS-/- dams, we treated mice with high
salt for 12 days before pregnancy until gestational day 18. Both AS+/+ and AS-/- mice
responded by increasing SBP suggesting that AS-/- dams also retained salt and fluid
even though plasma volume was not directly measured.
High salt diet had several interesting implications. Next to the blood pressure
lowering potency in pregnant wild type mice, it partially rescued the fetal phenotype
with a substantial effect on fetal weight, placental hypoxia, and placental efficiency in
both AS+/+ and
AS-/- dams. Placental efficiency can be altered experimentally by
manipulation of uterine blood flow, oxygen availability, and by the intake and
composition of maternal diet. Increased efficiency is also seen in naturally small
relative to large placentas in pigs, sheep, goats, rats and mice. Placental efficiency in
these polytocous species is positively related to litter size [132]. We found that
smaller placentas in AS+/+ dams on high salt diet and AS-/- dams were associated
with larger litter size indicating positive correlation between placental efficiency and
litter size. Decreased placenta weight with larger litter size in AS+/+ dams on high salt
diet also suggests increased placental efficiency thus supporting an unrestricted fetal
growth. While small, dark placentas were still observed in AS-/- dams on high salt diet
similar to control diet it remains amenable a consequence of altered local
aldosterone availability to support placental development.
96
Limitations of this model of aldosterone deficiency are the high renin and
angiotensin II levels which may trigger local dysregulatory events in pre-eclampsia.
In the absence of aldosterone synthase, deoxycorticosterone, corticosterone and
some 18-hydroxycorticosterone are still synthesized via 11 -hydroxylase activity.
Thus, the live-long inhibition of aldosterone synthesis in these animals potentially
allowing for alternative mineralocorticoid active steroid hormones might have
supplemented some extrarenal aldosterone effects or even enhanced the inhibition
of proliferation of trophoblasts via the activation of glucocorticoid receptors.
Aldosterone availability appears an important prerequisite to support normal
feto-placental development. AS-/- dams had a feto-placental phenotype with
decreased litter size, smaller placentas and fetuses, yet no overt pre-eclampsia. In
the absence of aldosterone, the fetal phenotype might be rescued in part by high salt
diet. In wild type mice, the high salt diet led to profound blood pressure reduction in
pregnancy which suggests an important role of plasma volume and placental
perfusion for maternal well-being. Thus, the absence of aldosterone cannot trigger
preeclampsia-like symptoms in mice, but might well contribute to the development of
pre-eclampsia-related intrauterine growth restriction while pre-eclampsia needs to be
triggered by additional factors. Thus, our study supports the importance of
aldosterone in placental function in vivo and a possible role for high salt
supplementation in pregnancies specifically associated with hypoaldosteronism or
reduced plasma volume expansion to maintain feto-placental integrity. In contrast, in
pre-eclampsia, the benefits of salt supplementation are still controversial and
appears have no major impact on outcome as indicated by a recent meta-analysis.
97
6 PERSPECTIVES
Aldosterone availability appears to be an important prerequisite to support normal
feto-placental development. This hypothesis is based on the observations that AS-/dams had a feto-placental phenotype with decreased litter size, smaller placentas
and fetuses, yet no overt pre-eclampsia. In the absence of aldosterone, the fetal
phenotype might be rescued in part by the restoration of a higher plasma volume by
the addition of a high salt diet. In wild type mice, the addition of a high salt diet led to
profound blood pressure reduction in pregnancy which suggests an important role of
plasma volume and placental perfusion for maternal well-being. Thus, the absence
of aldosterone cannot trigger pre-eclampsia-like symptoms in mice, but might well
contribute to the development of pre-eclampsia-related intrauterine growth restriction
while pre-eclampsia needs to be triggered by additional factors. These data support
an important role of aldosterone in the placental development as suggested by
smaller weight and sized placentas, and necrotic and inflammatory placentas in AS-/dams. Thus, our study supports the importance of aldosterone in placental function
in vivo and a possible role for high salt supplementation in pregnancies specifically
associated with hypoaldosteronism or reduced plasma volume expansion to maintain
the feto-placental integrity. Aldosterone deficiency aids to explain reduced placental
size and decreased fetal weight in pregnancy disorders such as intrauterine growth
restriction and pre-eclampsia.
98
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ACKNOWLEDGEMENTS
I would like to thank:
- Prof. Carsten Wagner and Prof. Johannes Loffing, my thesis supervisors and
bosses for giving me the opportunity to work in their groups, their valuable guidance
and support throughout the years of my thesis;
- Prof. François Verrey and Prof. Dominique Eladari, members of my thesis
committee, for valuable suggestions and advice concerning my project;
- All the people who contributed to the project with their work and expertise- Nicolas
Picard, Dominique Loffing-Cueni, Mariana Di Chiara, Mads Sorensen, Monique
Carrel, Nicole Kampik, Carla Bettoni, Marija Mihailova;
- Our collaborators Prof. Christoph Korbmacher and Viatcheslav Nesterov; Prof.
Felix Frey and Bernhard Dick for their work and expertise contributing to this project;
- Prof. Gerhard Rogler, Isabelle Frey-Wagner and Yu Wang for supply of few primers
and probes
- All the people working on J-floor in the Institutes of Physiology and Anatomy for
creating great working atmosphere;
- My parents (Pandurang and Jyotsna Todkar), Amit and Aparna for their great
support during all the years of my studies and my PhD;
- My wife Prajakta for her love and especially for her patience during tough time of
my PhD;
- My cousine Kiran and friends Pankaj, Nikhil, Manoj, Dheeraj, Pallavi, Samyuktha
and Alok for making my life easier in Zurich during PhD;
Further I would like to thank:
- Zurich Center for Integrative Human Physiology “ZIHP”, for the PhD student
fellowship.
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CURRICULUM VITAE
Name
Nationality
Marital status
Date of Birth
E-mail
Mobile
Abhijeet Pandurang Todkar
Indian
Married
20th January 1980
[email protected]
(041) 789299135
EDUCATION
Since Oct 2007
PhD fellow, Institute of Physiology and Institute of
Anatomy),
University of Zurich, Switzerland
Title of Projects: 1.Aldosterone dependent and
independent regulation of potassium homeostasis.
2. Role of aldosterone in pregnancy
Project advisors: Prof. Carsten Wagner and Prof.
Johannes Loffing
Jan 2006 to Aug 2007
M.Sc. Molecular Biology, University of Skovde,
Sweden and Eberhard Kares University of
Tübingen, Germany
Title of thesis: Transdifferentiation of vascular smooth
muscle cells.
Project Advisor: Prof. Robert Feil, (Department of
Signal Transduction-Transgene Models)
Jan 2003 to Feb 2005
Master of Veterinary Science (M.V.Sc.) - Veterinary
Pathology.
Bombay Veterinary College (BVC), India.
Title of thesis: Pathology of Streptozotocin (STZ)
induced hyperglycemia in rat and effect of herbal
medicine.
Project advisor: Prof. P.L. Lonkar
Sept 2007- Sept 2002
Bachelor of Veterinary science (B.V. Sc.)
K.N.P. College of veterinary Science, Shirwal Satara
MAFSU University, Satara
110
PUBLICATIONS
Papers
1. Abhijeet Todkar, Marianna Di Chiara, Dominique Loffing-Cueni, Carla Bettoni,
Markus Mohaupt, Johannes Loffing, Carsten A Wagner. Aldosterone deficiency
adversely affects pregnancy outcome in mice. Pflügers Arch, in press
2. Abhijeet Todkar, Nicolas Picard, Dominique Loffing-Cueni, Mads Sorensen,
Marija Mihailova, Christoph Korbmacher, Natalia Makhanova, Oliver Smithies,
Carsten Wagner, Johannes Loffing. Aldosterone is dispensable for renal but not for
colonic regulation of potassium homeostasis. (In preparation).
Abstracts (Poster and Oral Presentations)
Abhijeet Todkar, Nicolas Picard, Dominique Loffing-Cueni, Marija Mihailova,
Carsten A. Wagner, Johannes Loffing. Expression of Na+-Cl- cotransporter (NCC)
and Cl-/anion exchanger pendrin in aldosterone-deficient mice. Poster presented in
Transporter 2008 meeting, Murten, August 2008, Switzerland.
Abhijeet Todkar, Jana Kovacikova, Marija Mihailova, Natalia Makhanova, Olivier
Smithies, Johannes Loffing, Carsten A Wagner. Acidosis in mice lacking aldosterone
synthase. 5th symposium of ZHIP (Zurich Center for integrative human Physiology),
30 August 2009, University Hospital, Zurich, Switzerland.
Abhijeet Todkar, Jana Kovacikova, Marija Mihailova, Natalia Makhanova, Olivier
Smithies, Johannes Loffing, Carsten A Wagner. Acidosis in mice lacking aldosterone
synthase.ISN Forefronts Symposium on Renal and Extrarenal Control of Acidbase Homeostasis in Health and Disease, 17 - 20 September 2009, Florence, Italy.
Abhijeet Todkar, Jana Kovacikova, Marija Mihailova, Natalia Makhanova, Olivier
Smithies, Johannes Loffing, Carsten A Wagner. Acidosis in mice lacking aldosterone
synthase.American Society of Nephrology (ASN) Renal Week, 27.10 to 1.11. 2009,
San Diego, CA, USA.
Abhijeet Todkar, Jana Kovacikova, Marija Mihailova, Natalia Makhanova, Olivier
Smithies, Johannes Loffing, Carsten A Wagner. Acidosis in mice lacking aldosterone
synthase.Joint Meeting of the Scandinavian and German Physiological Societies, 2730 September, 2010, Copenhagen, Denmark.
Abhijeet Todkar, N Picard, D Loffing-Cueni, M Mihailova, N Makhanova3, O
Smithies, CA Wagner, J Loffing. Aldosterone-dependent and -independent regulation
of renal potassium excretion. Poster presented in 4th symposium of ZHIP (Zurich
111
Center for Integrative Human Physiology), 22 August 2008, University Hospital,
Zurich, Switzerland.
A Todkar, M Di Chiara, D Loffing-Cueni, C Bettoni, N Makhanova, O Smithies, J
Loffing, CA Wagner. Aldosterone deficiency during pregnancy does not lead to
preeclampsia but results in placenta dysfunction, reduced litter size, and smaller
pups. Poster presented in 7th International Symposium on Aldosterone and the
ENaC/Degenerin Family of Ion Channels: Molecular Mechanisms and
Pathophysiology, September 18-22, 2011. Pacific Grove, CA.
Abhijeet Todkar, Nicolas Picard, Mads Sorensen, Natalia Makhanova,
OliverSmithies, Carsten Wagner, Johannes Loffing. Aldosterone is diepensable for
renal but not for colonic regulation of potassium homeostasis. Oral presentation in
7th International Symposium on Aldosterone and the ENaC/Degenerin Family of Ion
Channels: Molecular Mechanisms and Pathophysiology, September 18-22, 2011.
Pacific Grove, CA.
A. Todkar, M. Di Chiara, D. Loffing-Cueni, C. Bettoni, M. G. Mohaupt, J. Loffing, C.
Wagner. Aldosterone deficiency adversely affects pregnancy outcome in mice.
Accepted for oral presentation in XVIII International Society for the Study of
Hypertension in Pregnancy (ISSHP) World Congress, 9th to 12th July, 2012, Geneva,
Switzerland.
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